The ubiquitin variant UbKEKS modifies PCNA to enhance DNA polymerase processivity during replication

The ubiquitin variant UbKEKS modifies PCNA to enhance DNA polymerase processivity during replication | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The ubiquitin variant UbKEKS modifies PCNA to enhance DNA polymerase processivity during replication Francois-Michel Boisvert, Julie Frion, Jennifer Raisch, Dominique Lévesque, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6515396/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Proliferating Cell Nuclear Antigen (PCNA) is a pivotal regulator of DNA replication and repair, orchestrating protein recruitment through its binding domains and post-translational modifications (PTMs). Lysine 164 (K164) of PCNA serves as a critical molecular switch modulated by ubiquitin (Ub) and ubiquitin-like proteins. Here, we identify the ubiquitin variant Ub KEKS as a new PCNA regulator, demonstrating its covalent modification of K164 and quantifying the Ub KEKS -to-Ub modification ratio. Loss of Ub KEKS results in delayed S-phase progression without altering PCNA levels or nuclear localization. However, DNA fiber combing assays reveal a significant reduction in DNA polymerase processivity in Ub KEKS -deficient cells, indicating its role in replication efficiency. At a whole-cell scale, the mapping by mass spectrometry of diGly remnants after trypsin digestion, demonstrate that modifications by Ub KEKS or Ub do not compensate one another, due to key differences regarding amino acids surrounding modified lysines. Ultimately, our findings establish Ub KEKS as a distinct key modulator of PCNA function, expanding the repertoire of PTMs that influence DNA replication dynamics. These insights pave the way for further exploration of Ub KEKS as a regulator of genome stability and cell cycle regulation. Biological sciences/Cell biology/Post-translational modifications/Ubiquitylation Biological sciences/Cell biology/Cell division/DNA replication/DNA synthesis Biological sciences/Biochemistry/Proteomics/Protein–protein interaction networks Biological sciences/Biological techniques/Proteomic analysis Biological sciences/Cell biology/Cell division/Checkpoints UbKEKS Proliferating Cell Nuclear Antigen (PCNA) Post-Translational Modification (PTM) DNA fiber combing assay Data-Independent Acquisition (DIA) Data-Dependent Acquisition (DDA) SILAC labelling PRM quantification AviTag Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Proliferating Cell Nuclear Antigen (PCNA) is a highly conserved nuclear protein that plays a central role in DNA replication and repair 1 , 2 . In humans, three PCNA proteins can assemble to form a homotrimer ring around DNA strands and serve as a recruitment platform for many cellular protagonists 1 , 3 – 6 . Nicknamed the maestro of replication forks, PCNA intervenes in many mechanisms such as origin firing 3 , 4 , 7 , increase of DNA polymerase processivity 3 , 4 , 8 , maturation of Okazaki fragments 3 , 4 , or even DNA damage tolerance through translesion synthesis (TLS) or error-free bypass 3 , 5 , 6 , 9 – 14 . Moreover, PCNA is involved in several DNA repair processes 3 – 6 , 15 – 20 and mechanisms of cell cycle regulation 3 , 4 , 14 , 21 . Coordination between all these machineries relies heavily on the modulation of a protein partner’s affinity for modified or unmodified PCNA 3 , 4 , 6 , 22 , 23 . Such affinity is primarily determined by the presence of PCNA binding motifs known as either PIP-box, degenerate PIP-box, APIM, specialized PIP-box for strong interaction, PIP-degron or inverted PIP-degron sequences, each resulting in different binding strengths 3 , 24 – 26 . Post-translational modifications (PTMs) also add an extra level of fine-tuning by granting the use of extra recognition sequences such as ubiquitin (Ub) or SUMO binding domains 3 , 6 . Therefore, combinations of PCNA-binding domains and PTMs reconnaissance sequences allow the creation of complex, yet precise modulations of PCNA interactome throughout the cell cycle. Many PTMs such as phosphorylation, acetylation, methylation or ubiquitination can modify one or several amino acids on PCNA 6 , 27 – 35 . Ub and other Ub-like proteins such as SUMO, ISG15 or NEDD8 have been shown to modify several lysine residues within PCNA 6 , 33 – 35 . In particular, the lysine 164 of PCNA serves as a central molecular switch in the mutually exclusive regulation of mechanisms by Ub and Ub-like modifiers. For instance, monoubiquitination of PCNA at lysine 164 by the E2 and E3 ubiquitin enzymes Ube2A-Rad18 triggers the recruitment of TLS polymerases at a damage site, hence initiating translesion synthesis 3 , 6 , 9 – 12 , 36 . On the other hand, addition of a NEDD8 protein by the E2 and E3 ubiquitin enzymes UBC12-Rad18 on PCNA’s lysine 164 will strongly reduce TLS polymerase recruitment at the lesion site 6 , 37 . Moreover, while polyubiquitination by a K63-type Ub chain of PCNA’s lysine 164 induces the recruitment of translocase ZRANB3 for error-free bypass mechanism via fork reversal 3 , 5 , 6 , 10 , 11 , 38 , SUMOylation of the same lysine prevents DNA repair via homologous recombination by recruiting the anti-recombinase PARI 3 – 6 , 39 . Despite all these examples, some mechanisms triggered by Ub and Ub-like modifications on PCNA’s lysine 164 have yet to be fully characterized 6 . Besides the four genes encoding the canonical Ub protein (UBA52, RPS27A, UBB and UBC), new Ub variants, encoded by genes that were initially described as pseudogenes, have been discovered 40 , 41 . Traditionally, pseudogenes are defined as an altered copy of a parental gene and cannot generate a functional protein 42 . However, Ub KEKS is an example of ubiquitin variant generated by the UBBP4 pseudogene and can be described as a fully functional PTM 41 , 43 , 44 . Surprisingly, only four amino acid differences (Q2K, K33E, Q49K and N60S) are required to allow Ub KEKS to modify a large number of proteins which diverge from canonical Ub targets 41 . Among those identified proteins, PCNA has been found as one of the top targets for Ub KEKS modification under normal conditions 41 , suggesting that Ub KEKS is a new regulator of PCNA and therefore, may impact some of its functions. Here, we validated that PCNA is covalently modified by Ub KEKS . We showed that Ub KEKS mainly binds PCNA on its lysine 164 and is required to enhance DNA polymerase processivity during DNA replication. More, a large-scale analysis of Ub and Ub KEKS signature revealed that both PTMs do not compensate for one another and highlighted key differences regarding amino acids surrounding modified lysines. Results PCNA can be modified by both Ub and Ub KEKS . To identify Ub and Ub KEKS targets, the combination of minimal AviTag sequence 45 and wild-type BirA (BirA WT ) enzyme was chosen due to its high efficiency and site-specific biotin labelling capacity 45 – 47 . Here, wild-type HeLa cells were transfected with plasmids encoding for 3 distinct constructs: GFP as a reporter protein; BirA WT -Myc; and either AviTag-HA alone, AviTag-HA-Ub or AviTag-HA-Ub KEKS (Fig. 1 a, Supp Table 1). Sequences coding for GFP, BirA WT -Myc and AviTag-HA are separated by T2A sequences which code for self-cleaving peptides 48 , as illustrated in Fig. 1 a. These T2A sequences induce ribosomal skipping during translation, allowing the production of three distinct proteins from a single ORF on the mRNA. After 48h of overexpression and 24h of biotin labelling, biotinylated proteins (i.e., proteins modified by either AviTag-HA-Ub or AviTag-HA-Ub KEKS ) were purified with streptavidin beads and identified using Data Independent Acquisition (DIA) mass spectrometry. Correct cleavage of all 3 constructs and the biotinylation process were validated by western blot (Fig. 1 b). As expected, GFP and BirA WT -Myc resulted in a single band around 26.9 kDa and 38.8 kDa respectively. Many proteins were successfully modified by either AviTag-HA-Ub or AviTag-HA-Ub KEKS (Fig. 1 b, left panel) while also being biotinylated (Fig. 1 b, right panel). As expected from previous studies 41 , HA and streptavidin profiles widely differ between AviTag-HA-Ub and AviTag-HA-Ub KEKS ; strongly suggesting that Ub and Ub KEKS target different pools of proteins. Mass spectrometry analysis also heavily implies different targets for Ub and Ub KEKS since some proteins were significantly biotinylated with AviTag-HA-Ub, but not with AviTag-HA-Ub KEKS ; and vice versa (Fig. 1 c, Supp Table 2). Proteins from almost all subcellular compartments were found significantly modified by Ub or Ub KEKS (Supp Table 2). Some interesting targets include PCNA, some translation initiation factors, histones, ubiquitin-like proteins and enzymes involved in the ubiquitination cascade (Fig. 1 c, Supp Table 2). Interestingly, E1 ubiquitin-activating enzyme UBA1 and E2 ubiquitin-conjugating enzyme UBE2N or UBE2NL were significantly enriched in AviTag-HA-Ub and AviTag-HA-Ub KEKS ; hinting that, like Ub, Ub KEKS requires an E1-E2-E3 enzymatic cascade to form peptide bonds. Finally, PCNA was also identified as a prominent target of both Ub and Ub KEKS , as it was featured in the top 1% (Z-score = 3.13) and top 5% (Z-score = 1.87) of enriched proteins for AviTag-HA-Ub and AviTag-HA-Ub KEKS respectively (Fig. 1 c, Supp Fig. 1 , Supp Table 2). This finding was then confirmed by immunoblotting (Fig. 1 d, Supp Fig. 2 ). PCNA-GFP was overexpressed in wild-type HeLa cells along either a control plasmid (empty pcDNA 3.1), Ub-HA or Ub KEKS -HA, before being purified using GFP-trap beads. Signals detected with GFP antibodies in immunoprecipitated samples (Fig. 1 d, central panel) show a similar amount of modified PCNA either in presence of Ub-HA or Ub KEKS -HA. However, detection of those same signals using HA antibodies instead (Fig. 1 d, right panel) reveals that in this case, PCNA is preferentially modified by Ub KEKS rather by Ub. Ub KEKS modification sites on PCNA only partially overlap with canonical ubiquitination sites and mainly involve PCNA’s lysine 164. PCNA is post-translationally modified on multiple amino acids by ubiquitylation, sumoylation or even neddylation 6 , 49 . As Ub and Ub KEKS present similar secondary and tertiary structures 50 , 51 , PCNA’s ubiquitination sites which are indexed in the mUbiSiDa database were considered as potential modification sites for Ub KEKS (Fig. 2 a). In mammals, eight lysines were listed as modified by Ub in the mUbiSiDa database 49 : K13, K14, K77, K80, K110, K164, K248 and K254. To identify Ub KEKS binding site(s), these lysines were mutated to arginines prior to co-immunoprecipitation experiments (Fig. 2 b-e, Supp Table 3). As a starting point, we initially mutated the first four and the last four lysines (Fig. 2 a-c). When overexpressing multi-mutated PCNA forms along with HA-Ub (Fig. 2 b), a light loss of PCNA modification was observed when the first four lysines (K13, K14, K77 and K80) were invalidated (IP-GFP, middle and right panels). On the other hand, when the last four lysines (K110, K164, K248 and K254) were mutated, a significant loss of PCNA’s modification was detected (IP-GFP, middle and right panel). This suggests that although Ub is capable of modifying all lysines presented in Fig. 2 a, PCNA modification by Ub mostly occurs on its last four lysines. For Ub KEKS , however, overexpression of PCNA with its first four lysines mutated along with Ub KEKS -HA led to a smaller loss of modification (Fig. 2 c, IP-GFP, middle and right panel). Also, mutation of PCNA’s last four lysines induced a significant loss of modification by Ub KEKS , though to a lesser extent than the loss observed for Ub modification (IP-GFP, middle and right panel). For further investigations, emphasis was put on the last four modifiable lysines of PCNA: K110, K164, K248 and K254 (Fig. 2 d-e). When the last four lysines were mutated one by one, all configurations except K248 induced a significant loss of PCNA modification by Ub (Fig. 2 d, middle and right panels). As expected, and according to mUbiSiDa, Ub is therefore able to modify multiple lysines on PCNA. Regarding Ub KEKS , only the mutation of K164 induced a decrease in PCNA modifications (Fig. 2 e, middle and right panels), suggesting that Ub KEKS mainly modifies PCNA on this specific lysine. Altogether, these observations indicate that even with similar secondary and tertiary structures, Ub KEKS modifies PCNA in a specific way compared to canonical Ub, targeting primarily the lysine 164 of PCNA. Ub and Ub KEKS modification do not compensate one another on total cell proteome level. A wider approach was used to map all modifications sites for Ub KEKS and Ub by mass spectrometry, using double glycine (diGly) immunoprecipitation in wild-type and Ub KEKS knockout HeLa cells 41 (Fig. 3 a). Indeed, due to Ub and Ub KEKS C-terminal sequences, a diGly remnant is generated on modified lysine after trypsin digestion 52 . Here, wild-type HeLa cells and Ub KEKS knockout HeLa cells (2 different clones named HeLa 2.7 and HeLa 4.3) were labelled with SILAC, using Arg 0 Lys 0 and Arg 6 Lys 4 medium respectively. Both cell lines (HeLa WT with either HeLa 2.7 or HeLa 4.3) were lysed and mixed with a 1:1 ratio, followed by trypsin digestion. Modified proteins were then purified using K-ε-GG antibody-coupled beads and analyzed by mass spectrometry. A total of 1451 diGly signatures were identified (Supp Fig. 3 a), distributed as either a unique or multiple modification sites per protein. Distribution of SILAC ratio (Log 2 SILAC (Ub KEKS knockout/Wild-type)) detected for every diGly signature was plotted in Fig. 3 b. Log 2 SILAC (Ub KEKS knockout/Wild-type) values lower than − 1 indicate a significant loss of diGly signatures in Ub KEKS knockout cells (blue color), whereas values superior to + 1 suggest a significant gain of diGly signature (red color). An important shift to the left (log 2 (ratio) lower than − 1, blue color) can be observed in Ub KEKS knockout cells, indicating that Ub does not compensate for the loss of Ub KEKS , and possibly do not share the same modification sites in most cases. Therefore, the 655 modification sites identified in the blue area were considered as specifically modified by Ub KEKS . To further investigate what could explain this specificity, distribution of the number of modification sites specific to Ub KEKS per protein was determined (Supp Fig. 3 b). Our result demonstrated that, like Ub, Ub KEKS can modify a protein either as a mono-, a multi-mono- or a poly-modification, leading to unique or multiple binding sites. Next, the conservation of residues surrounding lysines modified by Ub KEKS was analyzed and compared to the environment of all human lysines listed in the Uniprot Database (Fig. 3 c). Modification windows considered for this analysis ranged from 15 residues before to 15 residues after each lysine of interest. Although this bioinformatics study reveals no consensus sequence predicting Ub KEKS -specific modification sites, a significant enrichment of lysines and methionines around residues modified by Ub KEKS was identified. Simultaneous PTMs: 1 Ub KEKS modifies PCNA for every 41 bound Ub under normal conditions. Using data from the diGly signature mapping, Ub and Ub KEKS modification sites were found on PCNA (Fig. 3 d). Two distinct sites were detected on lysine K164 and K248: none of which, however, presented a significant loss of diGly signatures in Ub KEKS knockout HeLa cells. These observations suggested that, unlike our previous results using overexpressing systems (Fig. 1 , Fig. 2 ), the modification of PCNA by low level of endogenous Ub KEKS 41 might be overshadowed by the excessive amount of endogenous canonical Ub. Therefore, PCNA’s post-translational modification by Ub and Ub KEKS was quantified by mass spectrometry, using Parallel Reaction Monitoring (PRM) assay (Fig. 4 ). Wild-type HeLa cells were transfected with PCNA-GFP along with either empty plasmid pcDNA3.1, Ub-HA plasmid or Ub KEKS -HA plasmid. After 48h of overexpression, cells were harvested and prepared for mass spectrometry. Following trypsin digestion, heavy labelled peptides (Fig. 4 a) were added at a final concentration of 1.66fmol/µl (Fig. 4 b, Supp Fig. 4 ). To measure the amount of Ub and Ub KEKS in each sample, only the most intense fragment ions (y8, y7, y6, y4, y3 and b3) were considered (Fig. 4 b). Ub KEKS -to-Ub ratios were calculated using the light to heavy peptide ratio and then adjusted to the concentration of spiked-in heavy peptides (Fig. 4 c). Measured endogenous Ub KEKS :Ub ratio modifying PCNA was equal to 0.02416, meaning 1 Ub KEKS protein for every 41 Ub proteins was found on PCNA. Interestingly, overexpression of Ub-HA or Ub KEKS -HA lead to an adjustment of PCNA’s modification ratio. When Ub-HA was in excess, Ub KEKS :Ub ratio went down to 1:158 whereas; when Ub KEKS was in excess, Ub KEKS :Ub ratio went up to 11:1. Therefore, on top of quantifying the Ub KEKS :Ub ratio bound to PCNA under normal conditions; these last two measurements underline the flexibility of this balance, suggesting a dynamic response based on different cellular conditions. Ub KEKS knockout cells present a proliferation delay attributable to S phase tardiness. When it comes to functional consequences of this new type of modification, Ub KEKS knockout cells showed a proliferation delay and an increase of apoptosis 41 , 43 . As PCNA plays a key role in DNA replication, DNA repair and cell cycle control 2 – 4 , 6 , 21 , S-phase duration and cell cycle distribution were analyzed by flow cytometry (Fig. 5 ). Exponentially growing wild-type and Ub KEKS knockout (clone 2.7 and clone 4.3) cells were labelled for 45 min with BrdU, before being incubated with unlabeled medium. Cells were harvested by trypsinisation at t = 0h, t = 4h and t = 8h after medium change (BrdU pulse assay), and fixed prior to antibody labelling and flow cytometry analysis (Fig. 5 a). Populations of cells in late S-phase and in new G1 phase were measured at t = 4h and t = 8h respectively (Fig. 5 a-b). A small but significant delay of the S phase progression was observed 4 hours after the end of BrdU labelling (Fig. 5 b, upper panel). This proliferation delay between wild-type and Ub KEKS knockout HeLa cells was found exacerbated 8 hours after the change of culture medium (Fig. 4 b, lower panel). Cell cycle distributions of wild-type and Ub KEKS knockout cells were also investigated to detect potential defects of cell cycle checkpoint (Fig. 5 c). Populations of cells in G0/G1, S and G2 phase were measured and surprisingly revealed no significant accumulation in either one of those phases (Fig. 5 d). Due to the many known functions of PCNA at the replication fork 3 , 4 , emphasis for future experiments was put on the S phase. S phase delay in absence of Ub KEKS cannot be explained by PCNA cellular level nor its recruitment to the replication fork. Among all of PCNA’s perturbations susceptible to delay S phase, cellular levels in wild-type and Ub KEKS knockout cells were first investigated (Fig. 6 a-b). Raw data from our lab’s published work 43 were reanalyzed to determine endogenous abundance of PCNA (Fig. 6 a). Briefly, proteins from exponentially growing wild-type and Ub KEKS knockout HeLa cells were identified by DIA mass spectrometry using a special spectral library which includes several human cell lines, including HeLa cells 43 . In both wild-type and Ub KEKS knockout cells, over 18 unique peptides were detected for PCNA, covering more than 75% of the protein (Fig. 6 a). No intensity difference was detected, indicating that PCNA levels remained constant across all tested cell lines. This observation was then confirmed by western blot (Fig. 6 b). The second potential perturbation of PCNA to be tested was its sub-nuclear localization (Fig. 6 c). Indeed, PCNA is known to display a peculiar dynamic during S phase reflecting its different involvement during DNA replication (Supp Fig. 5 a 53 , 54 ). PCNA nuclear locations detected during all phases of the cell cycle are available in Supp Fig. 5 b. During S phase, PCNA was found under its DNA-bound form (Fig. 6 c). In wild-type HeLa cells, PCNA formed a dotted pattern in early S phase, a dotted ring pattern in mid-S phase and finally a speckled pattern in late S phase, confirming its dynamics previously reported in the literature. No difference in PCNA location was found in Ub KEKS knockout cells for any of the S phase steps, suggesting that PCNA’s recruitment to the replication fork is not impacted by Ub KEKS . Absence of Ub KEKS significantly decreases the processivity of DNA polymerases. Another well-known function of PCNA consists in increasing DNA polymerases processivity in proliferating cells 3 , 4 , 8 . To access DNA polymerase processivity, forks symmetry and replication structures present on ongoing forks, a DNA combing assay was performed in wild-type and Ub KEKS knockout cells (Fig. 6 d-g and Supp Fig. 6 ), using a successive labeling of CldU (red) and IdU (green) under normal conditions. Origin firing, elongating forks and colliding forks (termination) were identified and classified according to the chart presented in Supp Fig. 6 a. Ub KEKS knockout cells presented less initiation and more terminations events than wild-type cells (Supp Fig. 6 b). Significance of those biological observations was assessed using a Chi-square test (Supp Fig. 6 c), coupled with an additional Cramér’s V test to detect potential bias due to the large size of tested populations (over 650 measurements). Obtained V values were equal to 0.129, indicating that significant p-values calculated by the Chi-square were more likely due to the dataset’s size rather than a true biological difference. Next, DNA polymerase processivity was analyzed: Ub KEKS knockout cells displayed a striking delay in nucleotide incorporation compared to wild-type HeLa cells (Fig. 6 d-e). While DNA polymerases in wild-type cells presented an average replication speed of 1.11 kb/min, the ones in Ub KEKS knockout cells had their processivity almost cut in half (0.72 kb/min and 0.53 kb/min for HeLa 2.7 and HeLa 4.3 respectively). Since replication of the leading and lagging strands involves different DNA polymerases, each with their own replication speed 4 , 8 , the symmetry of elongating fork was also measured by dividing the length of the longer branch (if any) and divided by the shorter branch (Fig. 6 f-g). Only replication forks featuring the origin site and the successive CldU and IdU labelling were considered in this analysis. Overall, Ub KEKS knockout cells presented a slight increase of fork asymmetry. This variation, however, was found significant only for one of the two clones: HeLa 4.3 (Fig. 6 g). Ultimately, all these observations showed that PCNA modification by Ub KEKS plays a role (direct or indirect) in the regulation of DNA polymerases processivity. However, whether Ub KEKS also contribute to the discrimination between leading and lagging strands remains to be confirmed. Discussion PCNA forms an homotrimer that binds to DNA and acts as a recruitment platform for proteins involved in various cellular processes, including origin firing 3 , 4 , 7 , increasing DNA polymerases processivity 3 , 4 , 8 , Okazaki fragments maturation 3 , 4 , DNA damage tolerance 3 , 5 , 6 , 9 – 14 and DNA repair 3 – 6 , 15 – 20 . The regulation of these pathways heavily relies on the competitive binding affinity between unmodified or post-translationally modified PCNA and its interacting partners 3 , 4 , 6 , 22 , 23 . PTMs such as Ub and Ub-like proteins play a central role in fine-tuning those interactions by introducing additional binding sites via recognition sequences 3 , 6 . Ub KEKS is a recently discovered PTM that can specifically modify proteins and has been implicated in nucleolar composition and apoptosis regulation 41 , 43 . Here, we demonstrate that PCNA is one of the major targets of Ub KEKS . Our findings identified lysine 164 as the primary modification site, a residue widely recognized as a critical molecular switch for PCNA-mediated pathway regulation 3 , 6 . While Ub and Ub KEKS modifications do not compensate for each other, we showed that K164 can be modified by either of them. These two modifications likely rely on competitive binding as over-expression of Ub KEKS shifted the ratio of Ub KEKS and Ub covalently bound to PCNA. To explore the functional consequences of this modification, we used Ub KEKS knockout cells, which exhibit a proliferation delay. 41 , 43 . Given PCNA’s crucial role in DNA replication 3 , 4 , 7 , 8 , we investigated S-phase progression in these cells. Our results revealed that Ub KEKS deficiency induces a significant S-phase delay without affecting the overall cell cycle distribution. This delay was not attributable to alterations in PCNA abundance or its subnuclear localization. Instead, we observed a marked reduction in DNA polymerases processivity, with replication fork speed halved in Ub KEKS knockout cells. These findings suggest an essential role for Ub KEKS in regulating replication fork dynamics. PCNA can be subject to multiple PTMs, including phosphorylation, acetylation, methylation or ubiquitination 6 , 27 – 35 . Using affinity purification via the AviTag system and co-immunoprecipitation against GFP-tagged proteins, we established that Ub KEKS is covalently attached to PCNA, adding a novel layer of regulation. Notably, Ub KEKS does not modify PCNA with the same preferences as compared with Ub, targeting mainly K164. This residue is essential for protein recruitment and serves as a molecular switch coordinating PCNA-mediated mechanisms 6 , 55 , 56 . Additionally, we detected simultaneous modifications of PCNA by two PTMs, with at least one being Ub KEKS . However, it remains unclear whether this dual modification involves two Ub KEKS monomers on different lysines, a combination of Ub KEKS and Ub, or whether it forms a polychain. Regarding the functional implications of Ub KEKS modification, multiple possibilities exist due to PCNA’s involvement in numerous cellular pathways 3 – 20 . Since PCNA plays a central role in DNA replication and repair, and Ub KEKS deficiency leads to a proliferation delay 41 , 43 , we focused on S-phase regulation. The delay was not linked to changes in PCNA abundance, aligning with previous findings that Ub KEKS does not target proteins for proteasomal degradation 41 . Furthermore, despite Ub KEKS influencing the subcellular localization of nuclear and nucleolar proteins 41 , 43 , PCNA localization during S-phase remained unaffected in Ub KEKS KO cells, suggesting that impaired PCNA recruitment to the replication fork does not account for the observed delay. Instead, our data indicate that Ub KEKS depletion significantly reduces DNA polymerase processivity. Normally, PCNA recruits and stabilizes DNA polymerases, enhancing their processivity by up to 100-fold 4 , 8 . PCNA interactors compete for recruitment, influenced by PCNA-binding motifs, PTM modifications, and proximity to specific factors 3 , 4 , 8 , 57 . Given this, Ub KEKS likely serves as a crucial new regulator of DNA polymerase recruitment and stabilization by functioning as a PTM and providing additional recognition sequences. Moreover, Ub KEKS depletion correlated with an increase in asymmetric replication forks. Proximity to regulatory proteins such as the CMG complex or RPA proteins, is known to differentiate leading from lagging strand synthesis, facilitating selective recruitment of DNA polymerases 57 . This raises the possibility that Ub KEKS plays a role in distinguishing DNA strands, either by modifying PCNA itself or by acting on another regulatory factor. Collectively, these findings highlight Ub KEKS as a key regulator of DNA replication. On a broader scale, our findings illustrate the emergence of an entirely new research avenue concerning ubiquitylation. Both Ub and Ub KEKS specifically target a wide array of proteins 41 , 43 . Analysis of PCNA modification sites and immunoprecipitation against diGly signatures confirmed that Ub and Ub KEKS target different residues and do not compensate for each other at the cellular level. Although no strong consensus sequence for Ub KEKS modification emerged, we nevertheless observed an enrichment of lysines and methionines around its target sites, which contrast with the lack of lysines, methionines, and cysteines around residues modified by Ub 58 , 59 . This inverse correlation in residue composition may be a distinguishing feature between Ub and Ub KEKS target sites. Additionally, the selection of modification sites is largely determined by steric constraints imposed by E2 and E3 enzymes involved in the modification cascade 60 , 61 . Like Ub and other Ub-like proteins, Ub KEKS likely employs an enzymatic conjugation system, as suggested by the significant enrichment of E1 enzyme UBA1 and E2 enzyme UBE2N/UBE2NL in our AviTag pulldowns. This Ub KEKS enzymatic cascade, however, potentially involve distinct combinations E2 and E3 enzymes. Overall, our findings define Ub KEKS as a novel post-translational regulator of PCNA, adding a new dimension to its already intricate PTM-mediated regulation. Ub KEKS plays a critical role in DNA replication by enhancing DNA polymerase processivity. More broadly, Ub KEKS emerges as a unique PTM, distinct from Ub, targeting different proteins, and potentially utilizing a separate enzymatic conjugation pathway. These insights pave the way for further exploration of Ub KEKS as a universal PTM and its implications in cellular regulation. Methods Cell culture HeLa cells were cultured under normal conditions (37°C with 5% CO 2 ), as adherent cells in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FB Essence and 100U/mL Penicillin/streptomycin. Identification of Ub targets with AviTag constructs Generation of plasmids. AviTag constructs were designed to simultaneously express several tagged proteins (Fig. 1 a – Supp Table 1). Plasmids creation required two main steps: i) generation of the basic construction called pDONR-AviTag and ii) insertion of the sequences of the protein of interest (either tag HA alone, HA-tagged non-cleavable Ub 62 or HA-tagged Ub KEKS ). The non-cleavable version of Ub was used to maximize in-cellulo labelling of ubiquitinated proteins 62 . All sequences from the gene fragments used to create our plasmids are available in Supp Table 1. To create the pDONR-AviTag plasmid, a 1181 bp DNA gene fragment (Integrated DNA technologies, optimized sequence for mammalian cell expression) was designed to contain the following elements (Supp Table 1): an attb1 site followed by a KOZAK consensus sequence, the AviTag peptide sequence (GLNDIFEAQKIEWHE 45 ), a unique Kpn I restriction site, E. coli ’s BirA wt protein sequence with a C-terminal myc-tag sequence, a Glycine-Serine-Glycine (GSG) linker followed by a T2A self-cleaving peptide sequence 48 and, finally, an attb2 site. This gene fragment was used in a BP recombination reaction using BP Clonase II (Invitrogen #11789020) in combination with the attP -containing donor vector pDONR221 (Invitrogen #12536017) to generate the pDONR-AviTag plasmid. Next, three different gene fragments were ordered from Integrated DNA technologies as future inserts (Supp Table 1). These gene fragments contained a Gibson assembly 5’-end pairing sequence, a Kpn I restriction site, the HA tag sequence either alone, fused to Ub or fused to Ub KEKS DNA sequence, a GSG linker by a T2A self-cleaving peptide sequence as described before, a second Kpn I restriction site and, finally, a Gibson assembly 3’-end pairing sequence. Each gene fragment was used in a Gibson assembly reaction (New England Biolabs #M5510A) with the Kpn I-digested pDONR-AviTag plasmid. Resulting attL -containing pDONR constructs were then used in LR recombination reaction using LR Clonase II (Invitrogen #11791100) with either the attR -containing destination vector pDEST47 (Invitrogen #112281010) for HA tag alone and HA-Ub KEKS ; or the attR -containing destination vector pGLAP5.2 (gift from Peter Jackson, Addgene plasmid #19706). Obtained fusion proteins are summarized up in Supp Table 1. Biotin labelling and pulldown assay. Wild-type HeLa cells were transfected with either the control-AviTag, the Ub-AviTag or the Ub KEKS -AviTag plasmids (Supp Table 1). Cells were cultivated under normal conditions for 48h, then incubated with biotin for another 24h. Next, cells were washed with PBS 1X and scrapped in denaturing lysis buffer (8 M urea, 20 mM HEPES, 10 mM DTT). Samples were sonicated on ice for 1 min at 30% (Fischer Scientific Model 120 Sonic Dismembrator) with 5s on-5s off cycles. The lysates were then centrifuged at 4°C for 10 min at 16 000g to remove cellular debris. For each sample, 1 mg of total protein extract was incubated for 2h at room temperature with 25µl of high-performance streptavidin beads (Cytiva #17511301). Beads were then washed four times with lysis buffer before being transferred into low-bind tubes after the final wash. Finally, samples were prepared following the on-bead digestion protocol described below and send for Data Independent Acquisition (DIA) mass spectrometry. Confirmation of PCNA modification by Ub Cloning of the PCNA-GFP protein . mRNA was extracted from exponentially growing wild-type HeLa cells using TRIzol reagent (Invitrogen #15596026), according to the manufacturer’s instructions. PCNA sequence was amplified by RT-PCR with the primers PCNA_attb1 and PCNA_attb2, available in Supp Table 3. These oligos allowed to insert ATTB sites for Gateway cloning. A first recombination was performed from the PCR product into the pDONR221 plasmid (Invitrogen #12536017) using the BP Clonase II (Invitrogen #11789020), following the manufacturer’s protocol. Resulting plasmids were transformed into thermo-competent bacteria which were then seeded on LB plate with kanamycin and grown overnight. On the next morning, isolated colonies were harvested: plasmid constructions were extracted by mini-prep and validated by sequencing. Correct plasmids went under a LR recombination step into the pDEST47 plasmid (Invitrogen #112281010) using the LR Clonase II (Invitrogen #11791100). Plasmid final validations were done by sequencing. Protein overexpression and immunoprecipitation. Wild-type HeLa cells were cultivated in 6-wells plates until 60–70% confluency was reached. PCNA-GFP plasmid in combination with either the empty plasmid (pcDNA 3.1), HA-Ub or HA-Ub KEKS plasmid were co-transfected using Lipofectamine 2000 (Invitrogen #11668019), following the manufacturer’s guidelines and incubated for 48h. Cells were washed with PBS 1X and lysed in High Salt buffer (50 mM Tris pH 7.5, 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 1 mM DTT, 10% Glycerol, 1% NP40, PMSF 1X). Samples were sonicated on ice for 10s at 25% (Fischer Scientific Model 120 Sonic Dismembrator). The lysates were then centrifuged at 4°C for 10 min at 12 000g to remove cellular debris. 1 mg of each sample was incubated overnight at 4°C, with GFP-Trap agarose beads (ChromoTek, Martinsried, Germany). Beads were washed three times with High Salt buffer, before being incubated in Laemmli and boiled at 95°C for 5 min, to elute proteins. Samples were resolved on SDS-PAGE and transferred onto nitrocellulose membrane. Signals were revealed using GFP (Roche #11814460001, dilution 1:1000) and HA (Invitrogen #26183, dilution 1:1000) antibodies. Identification of modification sites on PCNA Choice of lysine according to pre-existing databases. Lysines potentially modified by Ub KEKS were selected based on the Ub modification sites listed in the mUbiSiDa website, a mammalian Ubiquitination Site Database 49 . Database was last accessed in April 2019. Eight lysines were chosen for mutated forms of PCNA: K13, K14, K77, K80, K110, K164, K248 and K254. Quadruple-mutated PCNA plasmids creation using gene fragments. Gene fragments containing the desired 4 mutations with attb1 and attb2 sites at each extremity were ordered from Integrated DNA Technologies (sequences available in Supp Table 3). As described in the previous section, final plasmids were generated using Gateway cloning technology. Gene fragments were mixed with pDONR221 plasmid (Invitrogen #12536017) and BP Clonase II (Invitrogen #11789020), and incubated at 25°C according to the manufacturer’s protocol. After transformation into thermo-competent cells and validation of the extracted plasmids by sequencing, correct products were incubated with pDEST47 plasmid (Invitrogen #112281010) and LR Clonase II (Invitrogen #11791100) following the manufacturer’s guidelines. Plasmid final validations were done by sequencing. Generation of single-mutation PCNA plasmid by site-directed mutagenesis. Plasmids containing mutated forms of PCNA were generated from the plasmid expressing wild-type PCNA-GFP, using site-directed mutagenic PCR as described by Liu et Naismith 63 . Briefly, 50 ng of DNA template was amplified by iProof DNA polymerase (BioRad #1725301), using both forward and reverse primers containing the desired mutation. Obtained PCR products were digested by DpnI for 1h, and the plasmids were transformed in DH10B thermocompetent E. coli bacteria (Thermo Scientific #EC0113). Plasmids were purified using a mini-prep kit (Favorgen Biotech Corp. #FAPDE-100) and final products were validated by enzymatic digestion and sequencing. Primers used to generate PCNA mutated plasmids are available in Supp Table 3. Overexpression and immunoprecipitation of mutated forms of PCNA. As described for the previous experiment, wild-type HeLa cells were cultured in 6-well plates until reaching 60–70% confluency. HA-Ub or HA-Ub KEKS plasmid along with a plasmid coding for different mutated forms of PCNA were co-transfected using Lipofectamine 2000 (Invitrogen #11668019). After 48h, cells were washed with PBS 1X and lysed in High Salt buffer (50 mM Tris pH 7.5, 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 1 mM DTT, 10% Glycerol, 1% NP40, PMSF 1X). Samples were sonicated on ice for 10s at 25% (Fischer Scientific Model 120 Sonic Dismembrator) and then centrifuged at 4°C for 10 min at 12 000g to remove cellular debris. 1 mg of each sample was incubated overnight at 4°C, with GFP-Trap agarose beads (ChromoTek, Martinsried, Germany). Beads were washed three times with High Salt buffer. Beads were then incubated in Laemmli and boiled at 95°C for 5 min, to elute proteins. Samples were resolved on SDS-PAGE and transferred onto nitrocellulose membrane. Loss of modification by Ub or by Ub KEKS was detected using GFP (Roche #11814460001, dilution 1:1000) and HA (Invitrogen #26183, dilution 1:1000) antibodies. Ub Knock-out HeLa cells HeLa cells invalidated for Ub KEKS were previously generated using the CRISPR/Cas9 method 41 . In this article, “HeLa 2.7” and “HeLa 4.3” refer to Ub KEKS knockout cells using two different combinations of sgRNAs to invalidate the UBBP4 pseudogene. Unless specified otherwise, these cells were also cultured as adherent cells and in the same conditions as those described in the previous paragraph on cell culture. Characterization of Ub and Ub modifications on PCNA by mass spectrometry SILAC labelling. Wild-type and Ub KEKS knockout HeLa cells were cultured in DMEM depleted of arginine and lysine (Life Technologies #A14431) which was supplemented with 10% triple dialyzed fetal bovine serum (Invitrogen #26400-044), 100U/mL Penicillin/streptomycin and 2 mM of GlutaMax (Gibco #35050-061). Lysines and arginines were added to create either light (Arg 0 Lys 0 , Sigma-Aldrich #A5006 and Sigma-Aldrich #L5501) or medium (Arg 6 Lys 4 , Cambridge Isotope Lab #CNM-2265 and Cambridge Isotope Lab # DLM-2640) labelled media. Final concentrations of each type of medium were 28µg/mL for arginine and 49µg/mL for lysine. L-proline was also added to reach a final concentration of 10 µg/mL and therefore, prevent unwanted arginine to proline conversion. Wild-type cells were labelled with light SILAC whereas knockout clones 2.7 and 4.3 were labelled in medium SILAC. Immunoprecipitation and preparation for mass spectrometry. Seven 150 mm plates of wild-type and Ub KEKS knockout cells were grown SILAC medium until confluency to obtain 20 mg of total cell extract. Proteins from each SILAC medium were mixed together to create two distinct solutions: either HeLa WT with HeLa 2.7 or HeLa WT with HeLa 4.3 (both solutions contained 1:1 ratio of SILAC labelled total extracts). Detection of Ub and Ub KEKS modification sites was made using the PTMScan® Pilot Ubiquitin Remnant Motif K-ε-GG kit (Cell Signaling Technology #14482)(Fig. 3 a): cell lysis, protein digestion, purification of lysate peptides and immunoaffinity purification of modified peptides were performed according to the manufacturer’s protocol. Final peptides obtained after all those steps were desalted and concentrated using ZipTips, prior being sent to the mass spectrometer for data-dependent acquisition (DDA). Analysis of the environment of Ub KEKS -modified lysines Python programming. Two short Python scripts were written to determine the number of appearances for each amino acid surrounding lysines of interest. The first script is designed for datasets containing truncated protein sequences measuring exactly 31 residues, with the lysine of interest at position 16. This type of data can typically be obtained using the diGly analysis featured in the MaxQuant 64 software for mass spectrometry experiments. The second script can be used on any dataset containing full protein sequences under a FASTA format. Both scripts rely on a string-based analysis and result in a final matrix indicating the number of appearances for each residue depending on their position within the lysine’s environment. Briefly, these scripts read the environment of each lysine given to the program by the user and identify the type of amino acid in each position. When a specific residue is identified within the surrounding of a lysine of interest, its associated counter in the final matrix is incremented. Once all lysines imputed by the user have been analyzed, the script returns the final matrix. Both Python scripts were made public on the GitHub repository at https://github.com/fmboisvert/KAnalysis , under the Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) license. For additional information and a short user manual, please refer directly to the files uploaded on GitHub. Applying the program to biological data. Python scripts described above were tested on 2 distinct datasets. On one hand, a list containing the residues sequences surrounding 655 lysines specifically modified by Ub KEKS were imputed into the first script. This list was obtained by selecting lysines presenting a significant drop of diGly motifs in Ub KEKS knockout cells (please refer to the paragraph entitled “Mass spectrometry data analysis” below). On the other hand, the Uniprot public database (20 386 entries, last accessed on October 27, 2021) was imputed into the second Python script to evaluate the average environment composition surrounding lysine in humans. This second analysis was defined as a control condition to compare the output of both scripts and therefore identify a potential enrichment or depletion of specific residues around modification sites specific to Ub KEKS . Quantification of the Ub KEKS :Ub ratio on PCNA by mass spectrometry Wild-type HeLa cells were cultured in 100 mm dishes under normal conditions until ~ 70% confluency. Cells were the co-transfected with a plasmid expressing PCNA-GFP along either a control plasmid (empty pcDNA3.1), a plasmid expressing Ub-HA or a plasmid expressing Ub KEKS -HA. Co-transfections were done using Lipofectamine 2000, according to the manufacturer’s instructions (Invitrogen #11668019). After 48h, cells were lysed in High Salt Buffer (50 mM Tris pH 7.5, 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 1 mM DTT, 10% Glycerol, 1% NP40, PMSF 1X, Roche Complete Protease Inhibitor Cocktail) and sonicated on ice for 10s at 35% (Fischer Scientific Model 120 Sonic Dismembrator). Lysates were then centrifuged at 4°C for 10 min at 12 000g. 1 mg of each sample was incubated with GFP-Trap agarose beads (ChromoTek, Martinsried, Germany) at 4°C, overnight. Beads were then washed 3 times with high-salt buffer and proteins were eluted using 2x Laemmli containing 10 mM DTT. Beads were boiled at 95°C for 5 min and let at room temperature to cooldown. Chloroacetamide (Sigma Aldrich #22790) was added to reach a final concentration of 50 mM and beads were incubated in the dark for 30 min. Following alkylation, samples were separated on a 4–12% gradient SDS-PAGE gel. Gel was the stained using SimplyBlue SafeStain (Invitrogen) and destained overnight in distilled water. Gel regions containing modified and unmodified PCNA-GFP were excised and proteins were digested in gel overnight using trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega Corporation, WI, USA). Peptides were then extracted from the gel pieces and a mix of unique heavy peptides were spiked into the samples at a final concentration of 1.66fmol/µl. This mix is composed of 4 heavy peptides with a 1:1:1:1 ratio: AQUA Ub (U-13C6, U-15N4; mass difference: +10 Da; sequence EGIPPDQQR – ThermoFischer Scientific), AQUA Ub KEKS (U-13C6, U-15N4; mass difference: +10 Da; sequence IQDEEGIPPDQQR - ThermoFischer Scientific), NEP Ub (U-13C6, U-15N; U-13C6, U-15N2; mass difference: +15 Da; sequence TLSDYNIQK - New England Peptide), NEP Ub KEKS (U-13C6, U-15N; U-13C6, U-15N2; mass difference: +15 Da; sequence TLSDYSIQK - New England Peptide). Then, the peptides were desalted using ZipTips C18 columns (EMD Millipore, Burlington, VT). Samples were dried by speed vac and resuspend in 1% formic acid. Peptides were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) at a wavelength of 205 nm. The peptides were then transferred into a glass vial (Thermo Fisher Scientific) and kept at − 20°C until analysis by mass spectrometry using PRM quantification. Study of the cell cycle of wild-type and knockout cells S phase progression monitoring. 200 000 wild-type and knockout cells were cultured in 6-wells plates under normal conditions until reaching 80% of confluency. Cells were labelled with warm medium containing 10µg of BrdU for 1h at 37°C, and then washed with warm PBS 1X, before warm medium without BrdU was added (BrdU chase). At 0h, 4h and 8h after the change of medium, cells were fixed in ethanol 70% at -20°C for at least 48h. Fixed cells were centrifuged and resuspended in a 2 N HCl and 0.5% of Triton X-100 solution. Samples were then incubated for 30 min at room temperature before being recentrifuged once again. Cell pellets were gently resuspended in NaB 4 O 7 0.1M (pH 8.5) and incubated for another 30 min at room temperature. Cells were centrifuged and pellets were resuspended in antibody solution (1% BSA, 0.2% Tween-20 in PBS 1X) containing mouse BrdU antibodies (BD Biosciences #347580, dilution 1:50). After a 30 min incubation at room temperature, cells were centrifuged and resuspended in antibody solution with anti-mouse Alexa Fluor 488 antibodies (Invitrogen, #A-11001, dilution 1:50), for 30 min at room temperature in the dark. Cells were centrifuged one last time and finally incubated in the dark for 30 min in a propidium iodide-RNAse solution (10 mg/mL propidium iodide, 0.25 mg/mL RNAse A in PBS 1X). Fluorophores signals were acquired by flow cytometry (BD Fortessa cytometer, Becton Dickinson) and analyzed with FlowJo, version 10.8.1 (Becton Dickinson & company). BrdU signal was plotted against propidium iodide labelling to precisely detect all cell cycle phases. Percentages of cells in late S phase at 4h and in new G1 phase at 8h were measured. For both time points, percentages of cells for each of the populations went under statistical analysis in GraphPad (GraphPad Software - version 9.0.0., USA). Cell cycle distribution analysis. The samples prepared for S-phase progression monitoring were also used to determine the cell cycle distribution in wild-type and Ub KEKS knockout cells. Percentages of cells in G1, S and G2/M phases went under statistical analysis in GraphPad (GraphPad Software - version 9.0.0., USA). Abundance of intracellular PCNA Analysis of mass spectrometry data comparing the total proteomes of wild-type and knockout cells. Mass spectrometry data generated by Data Independent Acquisition (DIA) 43 and available on the ProteomeXchange Consortium via the PRIDE 65 partner repository under the dataset identifier PXD040778; were analyzed by comparing whole cell proteomes from wild-type and Ub KEKS knockout cells. Data were loaded into the ProStaR software 66 (version 1.26.1). Identified proteins with a unique peptide count lower than 2 were filtered out and partially observed missing values were imputed with the slsa algorithm. Missing values for entire conditions were imputed using the Det quantile algorithm (Quantile = 1; Factor = 0,2). A differential abundance analysis was later performed in ProStaR to evaluate the impact of Ub KEKS on PCNA’s cellular levels. Comparison was made using the following parameters: sequence coverage, the number of unique peptides identified and the intensity detected for PCNA. Confirmation by western blot. Exponentially growing wild-type and Ub KEKS knockout cells were lysed in clear Laemmli buffer and sonicated on ice (Fischer Scientific Model 120 Sonic Dismembrator). Samples were centrifuged at 12 000g for 10 min (4°C) to get rid of cellular debris and 25µg of the supernatants were resolved by SDS-PAGE prior transfer on nitrocellulose membrane. Signals were detected with endogenous PCNA antibodies (Cell Signaling #2586S, dilution 1:1000) while ß-tubulin antibodies (Cell Signaling #2128S, dilution 1:1000) were used as loading control. Nuclear localization of PCNA during S-phase Wild-type and Ub KEKS knockout cells were seeded on glass coverslips in a 24-wells plate until 40% confluency. Cells were washed in PBS 1X and fixed for 15 min with 4% formaldehyde in PBS 1X at room temperature. Coverslips were washed twice with PBS 1X and cells were permeabilized for 10 min at -20°C, using ice-cold 100% methanol. Cells were washed again with PBS 1X prior to incubation for 1h in blocking buffer (5% Goat serum, 0.3% Triton X-100 in PBS 1X). Endogenous PCNA was labelled by using PCNA antibodies (Cell Signaling #2586S, dilution 1:2000) in blocking buffer (overnight incubation, 4°C). Cells were then washed three times with PBS 1X and incubated in blocking buffer containing goat anti-mouse Alexa Fluor 488 antibodies (Invitrogen, #A-11001, dilution 1:800) for 1h in the dark, at room temperature. Nuclei were stained with DAPI solution (1µg/µl) for 8 min in PBS 1X and cells were washed again twice. Coverslips were mounted on microscope slides using Immuno-mount medium (ThermoFischer Scientific). Images were acquired on a Zeiss LSM 880 confocal microscope using a 40× 1.4NA plan Apo objective. Image treatment and assembling were performed using Fiji (version 1.53c) software 67 . Image classification was based on peer-reviewed literature 53 , 54 . Nuclear locations of PCNA during S-phase are presented in Fig. 6 c while PCNA’s locations during other cell cycle phases are available in Supp Fig. 5 . Replication combing assay Exponentially growing wild-type and Ub KEKS knockout HeLa cells were cultured in a 6-wells plate until 80% confluency. Cells were labelled 45 min with 100µM of 5-chloro-2’-deoxyuridine (CldU, Sigma-Aldrich #C6891), followed by another incubation of 45 min with 100µM of 5-iodo-2’-deoxyuridine (IdU, Sigma-Aldrich #I7125). Cells were trypsinized and counted before being embedded into agarose plugs and going under DNA extraction following the protocol described in the FiberPrep kit (Genomic Vision, Bagneux, France). Coverslips coated with vinylsilane were combed with a constant 2 kb/µm stretching factor, using the molecular combing system FiberComb (Genomic Vision, Bagneux, France). Coverslips were baked for 2h at 60°C in the dark, then denatured and blocked with BlockAid solution (Invitrogen #B10710). DNA strands were labelled using mouse ssDNA (DSHB University of Iowa, United States), mouse BrdU (favors IdU labelling, BD Biosciences #347580), rat BrdU (favors CldU labelling, Abcam #AB6326) antibodies. After three PBS-Tween washes, secondary antibodies staining was performed with goat anti-mouse BV480 (BD Biosciences #564877), goat anti-mouse Alexa Fluor 555 (Invitrogen #A21424) and goat anti-rat Cy5 (Abcam, #AB6565). All antibodies dilutions were done following Genomic Vision’s protocol for replication combing assay. Coverslips were dehydrated by ethanol, mounted and pictures were acquired using the EasyScan service (Genomic Vision, Bagneux, France). DNA fibers were analyzed using the FiberStudio software. Fork speed for each cell line was calculated using the formula \(\:V\:=\:\left(fiber\:length\times\:\:stretching\:factor\right)÷labelling\:time\) . All statistics were performed using Graph Pad Prism version 9.0.0. (GraphPad Software, USA). To facilitate the observation of elongation forks, only signals corresponding to incorporated CldU (red) and incorporated IdU (green) are shown in this article. On-beads digestion of samples for mass spectrometry Beads bound to proteins of interest were washed five times with 20 mM NH 4 HCO 3 . After the final wash, beads were resuspended in 50µl of 10 mM DTT and 20 mM NH 4 HCO 3 , and incubated at 60°C for 30 min under agitation. Then, beads were left to cooldown before adding 50µl of 15 mM chloroacetamide (Sigma Aldrich #22790) in 20 mM NH 4 HCO 3 . Samples were incubated 1h in the dark at room temperature. DTT concentration in samples was raised to 15 mM to quench chloroacetamide. After a 10 min incubation at room temperature, 1µg of trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega Corporation, WI, USA) was added to the beads and tubes were incubated at 37°C overnight. Digestion was then stopped by acidifying the beads to a final concentration of 1% formic acid. Supernatants were collected and transferred into new lowbind tubes. Beads were resuspended in 60% acetonitrile and 1% formic acid solution before a 5 min incubation at room temperature. Supernatants were once again harvested and mix with their corresponding supernatants previously collected. Samples were dried by speedvac and desalted using ZipTips C18 column (EMD Millipore, Burlington, VT), and finally resuspended in 1% formic acid. Peptides were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) at a wavelength of 205 nm. The peptides were then transferred into a glass vial (Thermo Fisher Scientific) and kept at − 20°C until analysis by mass spectrometry. Mass spectrometry acquisition Data-independent acquisition (DIA). This method of acquisition was used to generate results presented in Fig. 1 c. 250 ng of peptides from each sample were injected into an HPLC (nanoElute, Bruker Daltonics) and loaded onto a trap column with a constant flow of 12 µl/min (Acclaim PepMap100 C18 column, 0.3 mm id x 5 mm, Dionex Corporation); then eluted onto an analytical C18 Column heated to 50°C (1.9 µm beads size, 75 µm x 25 cm, PepSep). Peptides were eluted over a 2-hour gradient of ACN (5–37%) in 0.1% FA at 400 nL/min while being injected into a TimsTOF Pro ion mobility mass spectrometer equipped with a Captive Spray nano electrospray source (Bruker Daltonics). Data were acquired using diaPASEF with a 100–1700 m/z mass range for TIMS-MS scan. For each single TIMS (100 ms) in diaPASEF mode, we used 1 mobility window consisting of 27 mass steps (m/z between 114 to 1414 with a mass width of 50 Da) per cycle (1.27 seconds duty cycle). These steps cover the diagonal scan line for + 2 and + 3 charged peptides in the m/z-ion mobility plane. Raw files were analyzed using DIA-NN 68 and the Uniprot human proteome database (version : March 21, 2020–75 776 entries). Further details are available in the Mass spectrometry data analysis paragraph. Data-dependent acquisition (DDA). This method of acquisition was used to generate results presented in Fig. 3 b- 3 c. Trypsin-digested peptides were loaded and separated onto a nanoHPLC system (Dionex Ultimate 3000). A total of 10 µl of the sample (1.5 µg) was first loaded with a constant flow of 4 µl/min onto a trap column (Acclaim PepMap100 C18 column, 0.3 mm id × 5 mm, Dionex Corporation). Peptides were then eluted off towards an analytical C18 column heated to 40°C (2 µm beads size, 75 µm × 50 cm, PepMap) with a linear gradient of 5–45% of solvent B (80% acetonitrile with 0.1% formic acid) over a 4 h gradient at a constant flow (200 nL/min). Peptides were then analyzed by an Orbitrap Q Exactive mass spectrometer (Thermo Scientific) using an EasySpray source at a voltage of 2.0 kV. Acquisition of the full scan MS survey spectra (m/z 350–1600) in profile mode was performed in the Orbitrap at a resolution of 70,000 using 1,000,000 ions. Peptides selected for fragmentation by collision-induced dissociation were based on the ten highest intensities for the peptide ions from the MS survey scan. The collision energy was set at 25% and resolution for the MS/MS was set at 17,500 for 500,000 ions with maximum filling times of 20 ms for the full scans and 60 ms for the MS/MS scans. All unassigned charge states as well as singly, seven and eight charged species for the precursor ions were rejected, and a dynamic exclusion list was set to 500 entries with a retention time of 40 s (10 ppm mass window). To improve the mass accuracy of survey scans, the lock mass option was enabled. Data acquisition was done using Xcalibur version 4.3.73.11. Identification and quantification of proteins identified by MS were done using the MaxQuant software version 1.5.2.8. 64 . Biological replicates were done three times and combined together for the MaxQuant analysis. PRM quantification of Ub KEKS : Ub modification of PCNA. This method of acquisition was used to generate results presented in Fig. 4 . Peptides were loaded and separated onto a nanoHPLC system (Dionex Ultimate 3000) with a constant flow of 4 µl/min onto a trap column (Acclaim PepMap100 C18 column, 0.3 mm id × 5 mm, Dionex Corporation, Sunnyvale, CA). Peptides were then eluted off towards an analytical C18 column heated to 40°C (2 µm beads size, 75 µm × 50 cm, PepMap) with a linear gradient of 5–45% of solvent B (80% acetonitrile with 0.1% formic acid) over a 42-min gradient at a constant flow (400 nL/min). Peptides were analyzed by an Orbitrap Q Exactive mass spectrometer (Thermo Scientific) using a Parallel Reaction Monitoring (PRM) method. An inclusion list containing the m/z values corresponding to the monoisotopic form of the heavy and light peptides of Ub (AQUA Ub :520.3/525.3 ; NEP Ub: 541.3/548.8) and Ub KEKS (AQUA Ub KEKS : 762.9/767.9 ; NEP Ub KEKS : 527.8/535.3) was generated. Acquisition of the MS spectra (with m/z from the inclusion list) was performed in the Orbitrap. The collision energy was set at 28% and resolution for the MS/MS was set at 35,000 for 200,000 ions with maximum filling times of 110 ms with an isolation width of 2.0 m/z. Data acquisition was done using Xcalibur version 4.3.73.11. and further analyzed on Skyline software 69 ( version 19.1.0.193). Mass spectrometry data analysis Identification of protein modified by Ub KEKS . Raw files were analyzed with DIA-NN and the Uniprot human proteome database (version: March 21, 2020, − 75 776 entries). Briefly, the following parameters were entered in DIA-NN 68 : 2 miscleavages were allowed; fixed modification was carbamidomethylation on cysteine; enzyme was set as Trypsin/P. A maximum of 2 variable modifications were defined: carbamylation on lysines and N-term extremities. A mass tolerance of 20 ppm was used both for precursor and fragment ions. Identification value "Precursor FDR" was set to 0.01. Other settings include Peptide length range: 7–30; Precursor charges range: 2–4; precursor m/z range: 300–1800; and finally, Fragment ion m/z range 200–1800. The option "Match between runs" was also allowed. Following peptide identification, data were filtered using Prostar 66 : only proteins found in at least two out of the three replicates were considered for further analysis. Contaminants were removed and a VSN normalization within conditions was applied. Missing values on entire condition (MEC) were imputed with minimal value (first quantile) and partial observed value (POV) were imputed with SLSA. Detected intensities were transformed using the log 2 function and Z-scores were calculated for each protein in all three datasets. Figures featuring log 2 intensity and Z-scores were plotted using GraphPad software (GraphPad Software - version 9.0.0., the USA). Mapping of Ub KEKS and Ub diGly signature. Analysis of raw MS data was done using MaxQuant (version 1.6.17.0) 64 . Quantification was done with light (Lys 0 Arg 0 ) and medium (Lys 4 Arg 6 ) labels, and considering a trypsin digestion of the peptides with no cleavages on lysine or arginine before a proline. A maximum of two missed cleavages were allowed with methionine oxidation and protein N-terminal acetylation as variable modifications of proteins and carbamidomethylation as fixed modification. The maximum number of modifications allowed per peptide was set to 5. Mass tolerance was set to a maximum of 7 ppm for the precursor ions and 20 ppm for the fragment ions. Re-quantification of selected isotopic patterns was allowed to obtain ratios of all SILAC pairs. Second peptide search was also allowed. The minimum length of peptides to be considered for quantification was set to seven amino acids and the false discovery rate threshold set to 5%. The minimum number of peptides to be used for the identification of proteins was set to one but only proteins identified with two or more peptides were considered in further analysis. Protein quantification was calculated using both unique and razor peptides. A table containing all diGly signature sites detected by mass spectrometry was also generated. This file was used to determine the Log 2 (M/L) SILAC ratio for each identified site in order to identify sites specifically modified by Ub KEKS . More, repartition of diGly sites per protein was analyzed for all detected sites, Ub KEKS specific sites and Ub specific sites. Finally, sequences of residues surrounding lysines specifically modified by Ub KEKS (i.e., log 2 (M/L) < -1) were also retrieved from the table given by the MaxQuant software. These sequences measuring exactly 31 amino acids were later used in a Python script to study the environment around lysines modified by Ub KEKS (please, refer to the paragraph “Analysis of the environment of Ub KEKS -modified lysines above). Ub KEKS : Ub equilibrium for PCNA’s modification. Identification and quantification of Ub and Ub KEKS peptides was performed on Skyline software 69 (version 19.1.0.193). For quantification, the most intense fragment ions were used for all peptides: y8, y7, y6, y4, y3 and b3 (Fig. 4 - Supp Fig. 4 ). The amount of Ub and Ub KEKS proteins were calculated using the light to heavy peptide ratio. Obtained ratios were plotted in GraphPad software (GraphPad Software - version 9.0.0., USA) for statistical analysis. Statistics Identification of proteins modified by Ub KEKS via the AviTag system. Detected intensities for each protein were transformed using the log 2 function to obtain datasets following Gaussian distributions. Z-score were then calculated for every data point. Proteins with a Z-score superior to the 90% percentile (0.9673 for CTL; 1.233 for Ub and 1.262 for Ub KEKS ) within each data set was considered as significantly enriched. Two additional thresholds were also considered: Z-scores higher than the 95% percentile (1.389 for CTL; 1.857 for Ub and 1.824 for Ub KEKS ) and Z-scores superior to the 99% percentile (2.085 for CTL; 3.018 for Ub and 2.965 for Ub KEKS ) (n = 4 independent experiments). Distribution of SILAC ratios associated to diGly signature sites. Significance thresholds were arbitrarily set for depletion or enrichment of at least 2-folds. On Fig. 3 b, these values correspond to log 2 (M/L) = -1 and log 2 (M/L) = + 1 (n = 3 independent experiments). Composition of the environment surrounding lysines modified by Ub KEKS . Enrichment (or depletion) of specific amino acid was calculated by comparing frequency distributions of residues surrounding lysines modified by Ub KEKS and surrounding all lysines listed in the Uniprot database (Fig. 3 c). Significance was determined by a Binomial test in Microsoft Excel with the following parameters: number of successes corresponds the occurrence of the residue in the Ub KEKS -modified lysines analysis; number of trials was set to 655; the probability was defined as proportion of the same residue at the same position found in the Uniprot’s lysines analysis; and finally cumulative was considered as true. A table containing of the calculated p-value for each amino acid and each position is available in Supp Table 5 (n = 3 independent experiments). Number of modification sites per protein. For all modification sites identified across the human proteome and the ones specifically targeted by Ub KEKS , the number of modification sites per protein is displayed in Supp Fig. 3 . The distribution between these 2 categories is similar and no further statistical analysis was performed (n = 3 independent experiments) Quantification of the Ub KEKS : Ub ratio modifying PCNA. For each combination of plasmids, mean and standard deviations of measured Ub KEKS :Ub ratios are the following: PCNA-GFP + pcDNA3.1 \(\:\:=\:2.416\times\:{10}^{-2}\:\pm\:0.011\) ; PCNA-GFP + Ub-HA \(\:\:=\:6.337\:\times\:\:{10}^{-3}\:\pm\:0.005\) ; et PCNA-GFP + Ub KEKS -HA \(\:\:=\:11.34\:\pm\:3.945\) . No further statistical test was performed (n = 3 independent experiments). S phase progression monitoring. HeLa WT’s population showed \(\:25.5\pm\:3.676\:\text{\%}\) of cells in late S phase at 4h and \(\:18.23\pm\:4.219\:\text{\%}\) of cells in G1 phase at 8h. On the other hand, population of HeLa 2.7 showed \(\:20.73\pm\:3.262\:\text{\%}\) of cells in late S phase at 4h and \(\:9.82\pm\:4.473\:\text{\%}\) of cells in G1 phase at 8h. Finally, HeLa 4.3 populations showed \(\:22.8\pm\:2.5\:\text{\%}\) of cells in late S phase at 4h and \(\:10.45\pm\:4.095\:\text{\%}\) of cells in G1 phase at 8h. Significance was determined by a Repeated Measures One-way ANOVA, followed by a Dunnett’s multiple comparisons post hoc test. At 4h, both comparison HeLa WT/HeLa 2.7 and HeLa WT/HeLa 4.3 revealed significant differences with p-values equal to 0.0025 and 0.0102 respectively. At 8h, these differences were exacerbated as p-values for both comparisons dropped below 0.0001 (n = 3 independent experiments). Cell cycle distribution analysis. Percentages of cells in G1, S and G2/M phases were measured and observed variations were assessed by a two-way ANOVA and Tukey’s multiple comparisons post hoc test. Comparison between HeLa WT and HeLa 2.7 cells resulted in nonsignificant p-values for all cell cycle phases (0.4744 for G0/G1 phase, 0.9561 for S phase and 0.6412 for G2/M phase). Comparison between HeLa WT and HeLa 4.3 cells led to the same conclusion (0.9782 for G0/G1 phase, 0.4307 for S phase and 0.8021 for G2/M phase)(n = 3 independent experiments). PCNA levels in total proteomes from wild-type and Ub KEKS knockout cells. Differential abundance was assessed with a Limma t-test with slim pi0 calibration. Molecular levels were considered as significantly modulated if at least a 2-fold difference was observed and the corresponding p-value was lower than or equal to 0.01. Differential analysis of HeLa WT with HeLa 2.7 on one hand, and HeLa WT with HeLa 4.3 on the other hand, resulted in p-values of \(\:3.11\times\:{10}^{-2}\) and \(\:2.61\times\:{10}^{-4}\) respectively. However, since all observed differences were lower than to 2-fold difference, variation of PCNA’s cellular levels were ruled out as not significant (n = 3 independent experiments, each read twice by the mass spectrometer). Processivity of DNA polymerases. Replication speed in wild-type HeLa cells was equal to \(\:1.11\pm\:\text{0,39}\) kb/min. Absence of Ub KEKS lead to replication speeds of \(\:0.72\pm\:\text{0,25}\) kb/min and \(\:0.53\pm\:\text{0,26}\) kb/min for HeLa 2.7 and HeLa 4.3 cells respectively. Significance was determined using a One-way ANOVA, with a Dunnett’s multiple comparison post hoc test. Both comparison between HeLa WT and each Ub KEKS knockout clone resulted in a p-value smaller than 0.001 (n > 150 measurements, distributed over 2 independent experiments). Symmetry of replication forks. A symmetrical ratio was determined for each cell line: \(\:1.29\:\pm\:0.4\) for HeLa WT; \(\:1.37\:\pm\:0.58\) for HeLa 2.7 and \(\:1.55\:\pm\:0.76\) for HeLa 4.3. Significance was determined by a Kruskal-Wallis test, followed by Dunn’s multiple comparisons assay. Comparisons HeLa WT/HeLa 2.7 and HeLa WT/HeLa 4.3 led to p-values equal to 0.2884 and 0.0156 respectively (n > 75 measures per samples, distributed over 2 independent experiments). Distribution of replication structures at active replication forks. Impacts of the presence or absence of Ub KEKS on the different replication structures were assessed using a Chi-square test. Since the analysis was performed on large populations, an additional Cramér’s V test was also done to evaluate potential bias. Parameters and results of both statistical tests are available in Supp Fig. 6 c (n = 782 for HeLa WT; 665 for HeLa 2.7 and 804 for HeLa 4.3; all distributed over 2 independent experiments). Declarations Acknowledgements J.F. is the recipient of a “Fonds de Recherche du Québec – Santé” (FRQS) studentship (grant number #313345). Funding was provided from the Canadian Institutes for Health Research, grant number #398925 to F.-M.B. F.-M.B. is a FRQS Senior scholar (award number 281824). X.R. is a recipient of a Canada Research Chair in Functional Proteomics and Discovery of Novel Proteins. X.R. and F.M.B. are members of the FRQS-funded “Centre de Recherche du CHUS”. Author contribution Conceptualization: J.F., X.R. and F-M.B.; Methodology: J.F., A.M. and F-M.B.; Formal analysis: J.F. and J.R.; Investigation: J.F., J.R., G.M., D.L. and M-L.D.; Programming: J.F.; Writing – original draft: J.F.; Writing – review and editing: J.F., X.R. and F-M.B.; Supervision: X.R. and F-M.B.; Project administration: F-M.B.; Funding acquisition: F-M.B. All authors have read and agreed to the final version of the manuscript. Competing interests The authors declare no competing interests. Materials and Correspondence Correspondence and material requests should be addressed to François-Michel Boisvert at [email protected] Images license Some figures used in this article were generated using the SMART medical art platform from Servier (https://smart.servier.com/). Raw images are licensed under a Creative Commons Attribution 3.0 Unported License. More information on this license is available at https://creativecommons.org/licenses/by/3.0/ Data availability The mass spectrometry proteomics data have been uploaded on the ProteomeXchange Consortium via the PRIDE 65 partner repository under the dataset identifier PXD061769. Code availability Python scripts used in this article are available in the GitHub repository at https://github.com/fmboisvert/KAnalysis, under the Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) license. References González-Magaña A, Blanco FJ (2020) Human PCNA structure, function and interactions. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6515396","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453756439,"identity":"1a135147-a9a8-4431-b8c9-c6badbf05136","order_by":0,"name":"Francois-Michel Boisvert","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3RMUvEMBTA8VcCueVJN4nkqF8hJYOLeF+lpXCz4CjUTr3l9NaI+F0CgboUXAvekFucHPIBCtqr4pTKud2Q//QI+fEIAQiFjrBZBRRAjHNkATQA2896mqD+JYSIf5AxQtlh5FRT667LfMNfm1vst8nF0/3OuvYS4pXfIc9mqRImf3wo6NtJ/S7n2xeZqm4JrM28ZMEzylFoKVoykMrkii2HE2eGB/oJfpNSLgZyg725+yGfIGL7FyGJQEIJUpOxkXQaBJvakq/2b0lYS+TZc21SNW9IqtoCWTe1pWis60uM19HOffTmnPE6sq65SuKNf8vw55X/HCfuh0KhUOiAvgBXhVa3Wk4nhAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8882-8619","institution":"Université de Sherbrooke","correspondingAuthor":true,"prefix":"","firstName":"Francois-Michel","middleName":"","lastName":"Boisvert","suffix":""},{"id":453756440,"identity":"eaca6f82-e6d9-4455-a71f-c4ed8210564a","order_by":1,"name":"Julie Frion","email":"","orcid":"https://orcid.org/0000-0001-9613-799X","institution":"Université de Sherbrooke","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Frion","suffix":""},{"id":453756441,"identity":"f0d62ba4-f2e0-4cc0-9c0b-f32b108cc295","order_by":2,"name":"Jennifer Raisch","email":"","orcid":"https://orcid.org/0000-0003-4393-5933","institution":"Université de Sherbrooke","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Raisch","suffix":""},{"id":453756442,"identity":"a1ed3202-8027-4a68-90d4-91bd855e2d9b","order_by":3,"name":"Dominique Lévesque","email":"","orcid":"https://orcid.org/0000-0001-7660-0457","institution":"Université de Sherbrooke","correspondingAuthor":false,"prefix":"","firstName":"Dominique","middleName":"","lastName":"Lévesque","suffix":""},{"id":453756443,"identity":"b49c787e-1cee-400c-aad0-43b62965ff92","order_by":4,"name":"Gwendoline Marbach","email":"","orcid":"https://orcid.org/0000-0002-2646-3121","institution":"Université de Sherbrooke","correspondingAuthor":false,"prefix":"","firstName":"Gwendoline","middleName":"","lastName":"Marbach","suffix":""},{"id":453756444,"identity":"3406fe62-0fcf-44c2-b2cd-a2dd4991efd1","order_by":5,"name":"Marie-Line Dubois","email":"","orcid":"","institution":"Université de Sherbrooke","correspondingAuthor":false,"prefix":"","firstName":"Marie-Line","middleName":"","lastName":"Dubois","suffix":""},{"id":453756445,"identity":"ac3ae963-ba6f-4a08-828a-b9eb2d9d02d0","order_by":6,"name":"Anna Meller","email":"","orcid":"","institution":"Université de Sherbrooke","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Meller","suffix":""},{"id":453756446,"identity":"5d5569ad-1cb4-409d-a2bf-89f436373813","order_by":7,"name":"Xavier Roucou","email":"","orcid":"https://orcid.org/0000-0001-9370-5584","institution":"Université de Sherbrooke","correspondingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"","lastName":"Roucou","suffix":""}],"badges":[],"createdAt":"2025-04-23 20:30:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6515396/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6515396/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82360123,"identity":"3bee251b-6146-46a3-a2cb-53b421e99371","added_by":"auto","created_at":"2025-05-09 11:37:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":618859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCNA can be modified by both Ub and Ub\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eKEKS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Identification of non-cleavable Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e targets using AviTag sequence and biotin labeling. Three distinct constructs were generated: AviTag-HA alone (control), AviTag-HA-Ub and AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e. They were overexpressed along with the BirA\u003csup\u003eWT\u003c/sup\u003e-Myc construct and a GFP reporter protein. All of these proteins are gathered in a single plasmid where their respective sequences are separated by T2A sequences, a self-cleaving peptide. After 48h of overexpression and addition of biotin, cells were lysed and biotinylated proteins (i.e., proteins modified by Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e) were purified using Streptavidin beads. Final samples were analyzed by Data Independent Acquisition (DIA) mass spectrometry. \u003cstrong\u003eb.\u003c/strong\u003e Validation of the AviTag system. Total cellular extracts were run on SDS-PAGE. Correct cleavage between each protein should result in a smear signal for AviTag-HA constructs (HA antibodies), a single band near 38.8 kDa for BirA\u003csup\u003eWT\u003c/sup\u003e-Myc (Myc antibodies), and a unique band of approximately 26.9 kDa for GFP alone. Biotinylation was also assessed using streptavidin-HRP coupled antibodies (n=4 independent experiments). \u003cstrong\u003ec.\u003c/strong\u003e Identification of some key proteins targeted by Ub\u003csup\u003eKEKS\u003c/sup\u003e or/and Ub. Z-scores were based on the log\u003csub\u003e2\u003c/sub\u003e of intensities detected by DIA mass spectrometry. Proteins with a Z-score higher than the 90% percentile were considered significantly modified by Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e (n=4 independent experiments). \u003cstrong\u003ed.\u003c/strong\u003e Validation of PCNA’s modifications by western blot. GFP-tagged PCNA was overexpressed in wild-type HeLa cells along with either nothing (pcDNA 3.1), HA-Ub or HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e. After a pulldown against GFP tag, PCNA modifications by Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e were detected using GFP and HA antibodies. The number of Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e monomers modifying PCNA is indicated by black arrows (n=3 independent experiments).\u003c/p\u003e","description":"","filename":"MainfiguresManuscript1.png","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/c2737198383bac4199d658a5.png"},{"id":82359306,"identity":"95f21d12-4c64-485c-8c01-dbdc2a513db5","added_by":"auto","created_at":"2025-05-09 11:29:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":278921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUb and Ub\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eKEKS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e target different sites on PCNA and both can modify PCNA’s lysine 164.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Lysines modifiable by Ub according to the mUbiSiDa database. These lysines were mutated into arginines to invalidate potential binding sites to PCNA. Quadruple mutated PCNA forms (PCNA\u003csup\u003eFirst 4K\u003c/sup\u003e and PCNA\u003csup\u003eLast 4K\u003c/sup\u003e) are indicated with their respective combinations of mutations. \u003cstrong\u003eb. and c.\u003c/strong\u003e Overexpression of wild-type and quadruple mutated forms of PCNA-GFP along with Ub-HA or Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA, respectively. After a pulldown against GFP tags, PCNA’s modifications by Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e were detected using GFP and HA antibodies. Numbers of Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e monomers covalently bound to PCNA are indicated on the right (n = 6 and n = 8 independent experiments, respectively). \u003cstrong\u003ed. and e.\u003c/strong\u003e Overexpression of wild-type and single mutated forms of PCNA-GFP along with Ub-HA or Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA, respectively. After a pulldown against GFP tags, PCNA’s modifications by Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e were detected using GFP and HA antibodies. Numbers of Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e monomers covalently bound to PCNA are indicated on the right (n = 4 and n = 6 independent experiments, respectively).\u003c/p\u003e","description":"","filename":"MainfiguresManuscript2.png","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/7e4c50ef8c1979a603c71ddf.png"},{"id":82359304,"identity":"f820336c-f04c-49c4-b7af-ecdb73eeb228","added_by":"auto","created_at":"2025-05-09 11:29:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177497,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMapping of remnants of Ub and Ub\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eKEKS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e across the human proteome and validation of PCNA’s modification sites by mass spectrometry.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e DiGly immunoprecipitation of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e modified peptides. Wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells were labelled in SILAC before being lysed and mixed using a 1:1 ratio. Samples were digested by trypsin to reveal diGly sites, a signature of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e modification. Modified peptides were then enriched by immunoprecipitation before being sent to the mass spectrometer for diGly remnants mapping. \u003cstrong\u003eb.\u003c/strong\u003e SILAC ratios of the modified peptides intensity measured by mass spectrometry. A log\u003csub\u003e2\u003c/sub\u003e SILAC ratio (HeLa KO/ HeLA WT) lower than -1 indicates a significant loss of diGly signatures in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells (hence a modification site specific to Ub\u003csup\u003eKEKS\u003c/sup\u003e). On the other hand, a log\u003csub\u003e2\u003c/sub\u003e SILAC ratio (HeLa KO/ HeLa WT) superior to +1 suggests a significant gain of diGly signatures in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells (n = 3 independent experiments for each cell line). \u003cstrong\u003ec.\u003c/strong\u003e Enrichment (red) or depletion (blue) of amino acids surrounding the modification sites specific to Ub\u003csup\u003eKEKS\u003c/sup\u003e. Frequency distribution of each amino acid was determined for positions surrounding lysines modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e (655 sites identified in Figure 3b) or surrounding all lysines listed in the Uniprot database. Enrichment was calculated by comparing both frequency distributions. Significance of this enrichment was determined using a Binomial test. A p-value smaller than 0.05 was considered significant (i.e., a -log\u003csub\u003e10\u003c/sub\u003e P(enrichment) superior to 1.3). All p-values for each position and each amino acid are available in Supplementary Table 5 (n = 3 independent experiments). \u003cstrong\u003ed.\u003c/strong\u003e DiGly sites identified on PCNA. Lysines 164 and 248 were found modified in both wild-type and knockout HeLa cells. Modified lysine is indicated by a star. Depletion of diGly sites in cells invalidated for Ub\u003csup\u003eKEKS\u003c/sup\u003e was found negligible (n = 3 independent experiments).\u003c/p\u003e","description":"","filename":"MainfiguresManuscript3.png","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/04163947aa61bbe938a7b691.png"},{"id":82359303,"identity":"d94d958b-9bb0-4fa0-8a6f-1558049400ee","added_by":"auto","created_at":"2025-05-09 11:29:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":155197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of the Ub\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eKEKS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-Ub balance modifying PCNA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Precursors and their resulting fragment ions used to quantify Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e modifying PCNA. \u003cstrong\u003eb.\u003c/strong\u003e Fragment ion chromatograms for Ub and Ub\u003csup\u003eKEKS \u003c/sup\u003ein wild-type HeLa cells overexpressing either PCNA-GFP + pcDNA3.1, PCNA-GFP + Ub-HA or PCNA-GFP + Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA. The red curve indicates the overall signal for endogenous peptides while the blue curve represents the overall signal measured for heavy peptides. Square areas featured on the ride side of each chromatogram provide a zoomed-in version of the detected peaks. A more detailed version, individually featuring all ions detected is available in Supplementary Figure 4. m/z values are indicated in the box above each chromatogram. Peak values with mass errors are displayed above each peak. Dotted black lines define which peak was considered for the quantification of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e modifications (n = 3 independent experiments). \u003cstrong\u003ec.\u003c/strong\u003e Ub\u003csup\u003eKEKS\u003c/sup\u003e:Ub ratio detected in PCNA pulldown assay. Means and standard deviations are displayed. An estimation of the ratio in terms of number of proteins rather than intensity is available above each bar (n = 3 independent experiments).\u003c/p\u003e","description":"","filename":"MainfiguresManuscript4.png","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/d54b38108117b34fed98fa36.png"},{"id":82360124,"identity":"3ac379e4-c819-4fff-b81a-275cb6f5407f","added_by":"auto","created_at":"2025-05-09 11:37:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":441192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbsence of Ub\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eKEKS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e induces a longer S phase without disrupting overall cell cycle distribution.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e BrdU pulse-chase assay for S phase progression monitoring. Exponentially growing wild-type and knockout HeLa cells were labelled with BrdU for 1h under normal conditions before having their medium replaced by fresh and non-labelled DMEM. Cells were harvested and fixed at the end of labelling (t = 0h), after 4h in non-labelled medium (t = 4h) and after 8h in no-labelled medium (t = 8h). BrdU incorporation was detected using specific antibodies and DNA content was colored with a solution of PI/RNAase. Single living cells were selected through successive gatings: final dot plots represent BrdU versus PI intensities, allowing the distinction between labelled and unlabelled cells progression through the cell cycle. Displayed percentages represent the mean of the population measured (n = 3 independent experiments).\u003cstrong\u003e b.\u003c/strong\u003e Percentage of cells in late S phase at 4h (top histogram) and in G1 phase at 8h (next cell cycle, bottom histogram). Population measurements were performed as illustrated in Figure 5a. Means and standard deviations are displayed. Significance of cell cycle delay was determined using a one-way ANOVA coupled with Dunnett’s multiple comparisons test. *, ** and **** respectively refers to a p-value equal to 0.0102, 0.0025 or smaller than 0.0001 (n = 3 independent experiments).\u003cstrong\u003e c.\u003c/strong\u003e Cell cycle profile by Propidium Iodide labelling. These representative plots were generated from the same data generated in the BrdU pulse experiment. Cell cycle’s phases are delimited by dotted lines (n = 3 independent experiments). \u003cstrong\u003ed.\u003c/strong\u003e Cell cycle overall distribution in presence or absence of Ub\u003csup\u003eKEKS\u003c/sup\u003e. Each population size was measured as shown in Figure 5c. Means and standard deviations are displayed. Significance was evaluated by a two-way ANOVA test, followed by Dunnett’s multiple comparisons test (n = 3 independent experiments).\u003c/p\u003e","description":"","filename":"MainfiguresManuscript5.png","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/2e3d2df2ee15791a065f49b1.png"},{"id":82359309,"identity":"287f6855-d841-417d-9ea0-88874cafaf05","added_by":"auto","created_at":"2025-05-09 11:29:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":721910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Cellular levels of PCNA in wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells. Peptides intensities were measured using Data Independent Acquisition (DIA) mass spectrometry. Significance was determined using a Limma test with a p-value cut-off of 0.01 (n = 3 independent experiments, each read twice by the mass spectrometer). \u003cstrong\u003eb.\u003c/strong\u003e Confirmation of PCNA’s cellular level by western blot. Endogenous PCNA levels were detected in total cell extracts of exponentially growing wild-type and knockout HeLa cells. ß-tubulin levels were used as a loading control (n = 3 independent experiments). \u003cstrong\u003ec.\u003c/strong\u003e PCNA nuclear location during S phase. PCNA is labelled in green while the nucleus is stained with DAPI. DNA-bound PCNA is specific to S phase and go from dotted in early S phase, to speckled in late S phase. Pictures for the entire cell cycle are available in Supplementary Figure 5b (n = 5 independent experiments). \u003cstrong\u003ed.\u003c/strong\u003e Representative pictures of elongating replication forks after successive incorporation of CldU (red) and IdU (green). Scale bar indicates 10µm (n \u0026gt; 150 measurements, distributed over 2 independent experiments). \u003cstrong\u003ee.\u003c/strong\u003e Replication speed of elongating forks detected in wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells. DNA polymerase processivity was calculated by converting the length of observed DNA fibers into kb units via a known stretching factor (2\u0026nbsp;kb/µm), and by then dividing by the labelling time. Means and standard deviations are indicated next to each sample’s name. Significance was determined using a One-way ANOVA, with a Dunnett’s multiple comparison post hoc test, “****” indicates an adjusted p-value lower than 0.001 (n \u0026gt; 150 measurements, distributed over 2 independent experiments). \u003cstrong\u003ef.\u003c/strong\u003e Evaluation of fork symmetry by dividing the length of the longer branch over the shorter one. A representative picture of symmetrical fork is displayed. Scale bar represents 10µm. \u003cstrong\u003eg. \u003c/strong\u003eFork symmetry in wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells. Means and standard deviations are indicated under each sample’s name. Significance was determined by a Kruskal-Wallis test, followed by Dunn’s multiple comparisons assay. * refers to a p-value equal to 0.0156 (n \u0026gt; 75 measures per samples, distributed over 2 independent experiments).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the mechanism contributing to the proliferation delay in absence of Ub\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eKEKS\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"MainfiguresManuscript6.png","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/2bc7a6b6e66c3b39e86c5295.png"},{"id":83989583,"identity":"3ec7ca4c-97e9-4126-938c-101e7a85b203","added_by":"auto","created_at":"2025-06-05 12:03:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4060554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/45263da4-ed19-4175-9628-a44e0237fb7d.pdf"},{"id":82359314,"identity":"f548b715-044c-442d-872e-9855c68a9b5b","added_by":"auto","created_at":"2025-05-09 11:29:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3521721,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6515396/v1/73940f7cf1071d0db75189e0.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The ubiquitin variant UbKEKS modifies PCNA to enhance DNA polymerase processivity during replication","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProliferating Cell Nuclear Antigen (PCNA) is a highly conserved nuclear protein that plays a central role in DNA replication and repair\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In humans, three PCNA proteins can assemble to form a homotrimer ring around DNA strands and serve as a recruitment platform for many cellular protagonists\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Nicknamed the maestro of replication forks, PCNA intervenes in many mechanisms such as origin firing\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, increase of DNA polymerase processivity\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, maturation of Okazaki fragments\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, or even DNA damage tolerance through translesion synthesis (TLS) or error-free bypass\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Moreover, PCNA is involved in several DNA repair processes\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and mechanisms of cell cycle regulation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Coordination between all these machineries relies heavily on the modulation of a protein partner\u0026rsquo;s affinity for modified or unmodified PCNA\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Such affinity is primarily determined by the presence of PCNA binding motifs known as either PIP-box, degenerate PIP-box, APIM, specialized PIP-box for strong interaction, PIP-degron or inverted PIP-degron sequences, each resulting in different binding strengths\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePost-translational modifications (PTMs) also add an extra level of fine-tuning by granting the use of extra recognition sequences such as ubiquitin (Ub) or SUMO binding domains\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, combinations of PCNA-binding domains and PTMs reconnaissance sequences allow the creation of complex, yet precise modulations of PCNA interactome throughout the cell cycle. Many PTMs such as phosphorylation, acetylation, methylation or ubiquitination can modify one or several amino acids on PCNA\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31 CR32 CR33 CR34\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Ub and other Ub-like proteins such as SUMO, ISG15 or NEDD8 have been shown to modify several lysine residues within PCNA\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In particular, the lysine 164 of PCNA serves as a central molecular switch in the mutually exclusive regulation of mechanisms by Ub and Ub-like modifiers. For instance, monoubiquitination of PCNA at lysine 164 by the E2 and E3 ubiquitin enzymes Ube2A-Rad18 triggers the recruitment of TLS polymerases at a damage site, hence initiating translesion synthesis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. On the other hand, addition of a NEDD8 protein by the E2 and E3 ubiquitin enzymes UBC12-Rad18 on PCNA\u0026rsquo;s lysine 164 will strongly reduce TLS polymerase recruitment at the lesion site\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Moreover, while polyubiquitination by a K63-type Ub chain of PCNA\u0026rsquo;s lysine 164 induces the recruitment of translocase ZRANB3 for error-free bypass mechanism via fork reversal\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, SUMOylation of the same lysine prevents DNA repair via homologous recombination by recruiting the anti-recombinase PARI\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Despite all these examples, some mechanisms triggered by Ub and Ub-like modifications on PCNA\u0026rsquo;s lysine 164 have yet to be fully characterized\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBesides the four genes encoding the canonical Ub protein (UBA52, RPS27A, UBB and UBC), new Ub variants, encoded by genes that were initially described as pseudogenes, have been discovered\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Traditionally, pseudogenes are defined as an altered copy of a parental gene and cannot generate a functional protein\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. However, Ub\u003csup\u003eKEKS\u003c/sup\u003e is an example of ubiquitin variant generated by the UBBP4 pseudogene and can be described as a fully functional PTM\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Surprisingly, only four amino acid differences (Q2K, K33E, Q49K and N60S) are required to allow Ub\u003csup\u003eKEKS\u003c/sup\u003e to modify a large number of proteins which diverge from canonical Ub targets\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Among those identified proteins, PCNA has been found as one of the top targets for Ub\u003csup\u003eKEKS\u003c/sup\u003e modification under normal conditions\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, suggesting that Ub\u003csup\u003eKEKS\u003c/sup\u003e is a new regulator of PCNA and therefore, may impact some of its functions.\u003c/p\u003e \u003cp\u003eHere, we validated that PCNA is covalently modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e. We showed that Ub\u003csup\u003eKEKS\u003c/sup\u003e mainly binds PCNA on its lysine 164 and is required to enhance DNA polymerase processivity during DNA replication. More, a large-scale analysis of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e signature revealed that both PTMs do not compensate for one another and highlighted key differences regarding amino acids surrounding modified lysines.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePCNA can be modified by both Ub and Ub\u003c/b\u003e \u003csup\u003e \u003cb\u003eKEKS\u003c/b\u003e \u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo identify Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e targets, the combination of minimal AviTag sequence\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and wild-type BirA (BirA\u003csup\u003eWT\u003c/sup\u003e) enzyme was chosen due to its high efficiency and site-specific biotin labelling capacity\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Here, wild-type HeLa cells were transfected with plasmids encoding for 3 distinct constructs: GFP as a reporter protein; BirA\u003csup\u003eWT\u003c/sup\u003e-Myc; and either AviTag-HA alone, AviTag-HA-Ub or AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Supp Table\u0026nbsp;1). Sequences coding for GFP, BirA\u003csup\u003eWT\u003c/sup\u003e-Myc and AviTag-HA are separated by T2A sequences which code for self-cleaving peptides\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. These T2A sequences induce ribosomal skipping during translation, allowing the production of three distinct proteins from a single ORF on the mRNA. After 48h of overexpression and 24h of biotin labelling, biotinylated proteins (i.e., proteins modified by either AviTag-HA-Ub or AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e) were purified with streptavidin beads and identified using Data Independent Acquisition (DIA) mass spectrometry. Correct cleavage of all 3 constructs and the biotinylation process were validated by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). As expected, GFP and BirA\u003csup\u003eWT\u003c/sup\u003e-Myc resulted in a single band around 26.9 kDa and 38.8 kDa respectively. Many proteins were successfully modified by either AviTag-HA-Ub or AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, left panel) while also being biotinylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, right panel). As expected from previous studies\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, HA and streptavidin profiles widely differ between AviTag-HA-Ub and AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e; strongly suggesting that Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e target different pools of proteins. Mass spectrometry analysis also heavily implies different targets for Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e since some proteins were significantly biotinylated with AviTag-HA-Ub, but not with AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e; and vice versa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Supp Table\u0026nbsp;2). Proteins from almost all subcellular compartments were found significantly modified by Ub or Ub\u003csup\u003eKEKS\u003c/sup\u003e (Supp Table\u0026nbsp;2). Some interesting targets include PCNA, some translation initiation factors, histones, ubiquitin-like proteins and enzymes involved in the ubiquitination cascade (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Supp Table\u0026nbsp;2). Interestingly, E1 ubiquitin-activating enzyme UBA1 and E2 ubiquitin-conjugating enzyme UBE2N or UBE2NL were significantly enriched in AviTag-HA-Ub and AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e; hinting that, like Ub, Ub\u003csup\u003eKEKS\u003c/sup\u003e requires an E1-E2-E3 enzymatic cascade to form peptide bonds. Finally, PCNA was also identified as a prominent target of both Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e, as it was featured in the top 1% (Z-score\u0026thinsp;=\u0026thinsp;3.13) and top 5% (Z-score\u0026thinsp;=\u0026thinsp;1.87) of enriched proteins for AviTag-HA-Ub and AviTag-HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supp Table\u0026nbsp;2). This finding was then confirmed by immunoblotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PCNA-GFP was overexpressed in wild-type HeLa cells along either a control plasmid (empty pcDNA 3.1), Ub-HA or Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA, before being purified using GFP-trap beads. Signals detected with GFP antibodies in immunoprecipitated samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, central panel) show a similar amount of modified PCNA either in presence of Ub-HA or Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA. However, detection of those same signals using HA antibodies instead (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, right panel) reveals that in this case, PCNA is preferentially modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e rather by Ub.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eUb\u003c/b\u003e \u003csup\u003e \u003cb\u003eKEKS\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emodification sites on PCNA only partially overlap with canonical ubiquitination sites and mainly involve PCNA\u0026rsquo;s lysine 164.\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePCNA is post-translationally modified on multiple amino acids by ubiquitylation, sumoylation or even neddylation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. As Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e present similar secondary and tertiary structures\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, PCNA\u0026rsquo;s ubiquitination sites which are indexed in the mUbiSiDa database were considered as potential modification sites for Ub\u003csup\u003eKEKS\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In mammals, eight lysines were listed as modified by Ub in the mUbiSiDa database\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e : K13, K14, K77, K80, K110, K164, K248 and K254. To identify Ub\u003csup\u003eKEKS\u003c/sup\u003e binding site(s), these lysines were mutated to arginines prior to co-immunoprecipitation experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e, Supp Table\u0026nbsp;3). As a starting point, we initially mutated the first four and the last four lysines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). When overexpressing multi-mutated PCNA forms along with HA-Ub (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), a light loss of PCNA modification was observed when the first four lysines (K13, K14, K77 and K80) were invalidated (IP-GFP, middle and right panels). On the other hand, when the last four lysines (K110, K164, K248 and K254) were mutated, a significant loss of PCNA\u0026rsquo;s modification was detected (IP-GFP, middle and right panel). This suggests that although Ub is capable of modifying all lysines presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, PCNA modification by Ub mostly occurs on its last four lysines. For Ub\u003csup\u003eKEKS\u003c/sup\u003e, however, overexpression of PCNA with its first four lysines mutated along with Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA led to a smaller loss of modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, IP-GFP, middle and right panel). Also, mutation of PCNA\u0026rsquo;s last four lysines induced a significant loss of modification by Ub\u003csup\u003eKEKS\u003c/sup\u003e, though to a lesser extent than the loss observed for Ub modification (IP-GFP, middle and right panel). For further investigations, emphasis was put on the last four modifiable lysines of PCNA: K110, K164, K248 and K254 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e). When the last four lysines were mutated one by one, all configurations except K248 induced a significant loss of PCNA modification by Ub (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, middle and right panels). As expected, and according to mUbiSiDa, Ub is therefore able to modify multiple lysines on PCNA. Regarding Ub\u003csup\u003eKEKS\u003c/sup\u003e, only the mutation of K164 induced a decrease in PCNA modifications (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, middle and right panels), suggesting that Ub\u003csup\u003eKEKS\u003c/sup\u003e mainly modifies PCNA on this specific lysine. Altogether, these observations indicate that even with similar secondary and tertiary structures, Ub\u003csup\u003eKEKS\u003c/sup\u003e modifies PCNA in a specific way compared to canonical Ub, targeting primarily the lysine 164 of PCNA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eUb and Ub\u003c/b\u003e \u003csup\u003e \u003cb\u003eKEKS\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emodification do not compensate one another on total cell proteome level.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA wider approach was used to map all modifications sites for Ub\u003csup\u003eKEKS\u003c/sup\u003e and Ub by mass spectrometry, using double glycine (diGly) immunoprecipitation in wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Indeed, due to Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e C-terminal sequences, a diGly remnant is generated on modified lysine after trypsin digestion\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Here, wild-type HeLa cells and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells (2 different clones named HeLa 2.7 and HeLa 4.3) were labelled with SILAC, using Arg\u003csub\u003e0\u003c/sub\u003eLys\u003csub\u003e0\u003c/sub\u003e and Arg\u003csub\u003e6\u003c/sub\u003eLys\u003csub\u003e4\u003c/sub\u003e medium respectively. Both cell lines (HeLa WT with either HeLa 2.7 or HeLa 4.3) were lysed and mixed with a 1:1 ratio, followed by trypsin digestion. Modified proteins were then purified using K-ε-GG antibody-coupled beads and analyzed by mass spectrometry. A total of 1451 diGly signatures were identified (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), distributed as either a unique or multiple modification sites per protein. Distribution of SILAC ratio (Log\u003csub\u003e2\u003c/sub\u003e SILAC (Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout/Wild-type)) detected for every diGly signature was plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. Log\u003csub\u003e2\u003c/sub\u003e SILAC (Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout/Wild-type) values lower than \u0026minus;\u0026thinsp;1 indicate a significant loss of diGly signatures in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells (blue color), whereas values superior to +\u0026thinsp;1 suggest a significant gain of diGly signature (red color). An important shift to the left (log\u003csub\u003e2\u003c/sub\u003e (ratio) lower than \u0026minus;\u0026thinsp;1, blue color) can be observed in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells, indicating that Ub does not compensate for the loss of Ub\u003csup\u003eKEKS\u003c/sup\u003e, and possibly do not share the same modification sites in most cases. Therefore, the 655 modification sites identified in the blue area were considered as specifically modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e. To further investigate what could explain this specificity, distribution of the number of modification sites specific to Ub\u003csup\u003eKEKS\u003c/sup\u003e per protein was determined (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Our result demonstrated that, like Ub, Ub\u003csup\u003eKEKS\u003c/sup\u003e can modify a protein either as a mono-, a multi-mono- or a poly-modification, leading to unique or multiple binding sites. Next, the conservation of residues surrounding lysines modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e was analyzed and compared to the environment of all human lysines listed in the Uniprot Database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Modification windows considered for this analysis ranged from 15 residues before to 15 residues after each lysine of interest. Although this bioinformatics study reveals no consensus sequence predicting Ub\u003csup\u003eKEKS\u003c/sup\u003e-specific modification sites, a significant enrichment of lysines and methionines around residues modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e was identified.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSimultaneous PTMs: 1 Ub\u003c/b\u003e \u003csup\u003e \u003cb\u003eKEKS\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emodifies PCNA for every 41 bound Ub under normal conditions.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing data from the diGly signature mapping, Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e modification sites were found on PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Two distinct sites were detected on lysine K164 and K248: none of which, however, presented a significant loss of diGly signatures in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells. These observations suggested that, unlike our previous results using overexpressing systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the modification of PCNA by low level of endogenous Ub\u003csup\u003eKEKS\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e might be overshadowed by the excessive amount of endogenous canonical Ub. Therefore, PCNA\u0026rsquo;s post-translational modification by Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e was quantified by mass spectrometry, using Parallel Reaction Monitoring (PRM) assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Wild-type HeLa cells were transfected with PCNA-GFP along with either empty plasmid pcDNA3.1, Ub-HA plasmid or Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA plasmid. After 48h of overexpression, cells were harvested and prepared for mass spectrometry. Following trypsin digestion, heavy labelled peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) were added at a final concentration of 1.66fmol/\u0026micro;l (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To measure the amount of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e in each sample, only the most intense fragment ions (y8, y7, y6, y4, y3 and b3) were considered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Ub\u003csup\u003eKEKS\u003c/sup\u003e-to-Ub ratios were calculated using the light to heavy peptide ratio and then adjusted to the concentration of spiked-in heavy peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Measured endogenous Ub\u003csup\u003eKEKS\u003c/sup\u003e:Ub ratio modifying PCNA was equal to 0.02416, meaning 1 Ub\u003csup\u003eKEKS\u003c/sup\u003e protein for every 41 Ub proteins was found on PCNA. Interestingly, overexpression of Ub-HA or Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA lead to an adjustment of PCNA\u0026rsquo;s modification ratio. When Ub-HA was in excess, Ub\u003csup\u003eKEKS\u003c/sup\u003e:Ub ratio went down to 1:158 whereas; when Ub\u003csup\u003eKEKS\u003c/sup\u003e was in excess, Ub\u003csup\u003eKEKS\u003c/sup\u003e:Ub ratio went up to 11:1. Therefore, on top of quantifying the Ub\u003csup\u003eKEKS\u003c/sup\u003e:Ub ratio bound to PCNA under normal conditions; these last two measurements underline the flexibility of this balance, suggesting a dynamic response based on different cellular conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eUb\u003c/b\u003e \u003csup\u003e \u003cb\u003eKEKS\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eknockout cells present a proliferation delay attributable to S phase tardiness.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhen it comes to functional consequences of this new type of modification, Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells showed a proliferation delay and an increase of apoptosis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. As PCNA plays a key role in DNA replication, DNA repair and cell cycle control\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, S-phase duration and cell cycle distribution were analyzed by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Exponentially growing wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout (clone 2.7 and clone 4.3) cells were labelled for 45 min with BrdU, before being incubated with unlabeled medium. Cells were harvested by trypsinisation at t\u0026thinsp;=\u0026thinsp;0h, t\u0026thinsp;=\u0026thinsp;4h and t\u0026thinsp;=\u0026thinsp;8h after medium change (BrdU pulse assay), and fixed prior to antibody labelling and flow cytometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Populations of cells in late S-phase and in new G1 phase were measured at t\u0026thinsp;=\u0026thinsp;4h and t\u0026thinsp;=\u0026thinsp;8h respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). A small but significant delay of the S phase progression was observed 4 hours after the end of BrdU labelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, upper panel). This proliferation delay between wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells was found exacerbated 8 hours after the change of culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, lower panel). Cell cycle distributions of wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells were also investigated to detect potential defects of cell cycle checkpoint (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Populations of cells in G0/G1, S and G2 phase were measured and surprisingly revealed no significant accumulation in either one of those phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Due to the many known functions of PCNA at the replication fork\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, emphasis for future experiments was put on the S phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eS phase delay in absence of Ub\u003c/b\u003e \u003csup\u003e \u003cb\u003eKEKS\u003c/b\u003e \u003c/sup\u003e \u003cb\u003ecannot be explained by PCNA cellular level nor its recruitment to the replication fork.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong all of PCNA\u0026rsquo;s perturbations susceptible to delay S phase, cellular levels in wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells were first investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). Raw data from our lab\u0026rsquo;s published work\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e were reanalyzed to determine endogenous abundance of PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Briefly, proteins from exponentially growing wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells were identified by DIA mass spectrometry using a special spectral library which includes several human cell lines, including HeLa cells\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In both wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells, over 18 unique peptides were detected for PCNA, covering more than 75% of the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). No intensity difference was detected, indicating that PCNA levels remained constant across all tested cell lines. This observation was then confirmed by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The second potential perturbation of PCNA to be tested was its sub-nuclear localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Indeed, PCNA is known to display a peculiar dynamic during S phase reflecting its different involvement during DNA replication (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e). PCNA nuclear locations detected during all phases of the cell cycle are available in Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb. During S phase, PCNA was found under its DNA-bound form (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). In wild-type HeLa cells, PCNA formed a dotted pattern in early S phase, a dotted ring pattern in mid-S phase and finally a speckled pattern in late S phase, confirming its dynamics previously reported in the literature. No difference in PCNA location was found in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells for any of the S phase steps, suggesting that PCNA\u0026rsquo;s recruitment to the replication fork is not impacted by Ub\u003csup\u003eKEKS\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAbsence of Ub\u003c/b\u003e \u003csup\u003e \u003cb\u003eKEKS\u003c/b\u003e \u003c/sup\u003e \u003cb\u003esignificantly decreases the processivity of DNA polymerases.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnother well-known function of PCNA consists in increasing DNA polymerases processivity in proliferating cells\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. To access DNA polymerase processivity, forks symmetry and replication structures present on ongoing forks, a DNA combing assay was performed in wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-g and Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), using a successive labeling of CldU (red) and IdU (green) under normal conditions. Origin firing, elongating forks and colliding forks (termination) were identified and classified according to the chart presented in Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells presented less initiation and more terminations events than wild-type cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Significance of those biological observations was assessed using a Chi-square test (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), coupled with an additional Cram\u0026eacute;r\u0026rsquo;s V test to detect potential bias due to the large size of tested populations (over 650 measurements). Obtained V values were equal to 0.129, indicating that significant p-values calculated by the Chi-square were more likely due to the dataset\u0026rsquo;s size rather than a true biological difference. Next, DNA polymerase processivity was analyzed: Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells displayed a striking delay in nucleotide incorporation compared to wild-type HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-e). While DNA polymerases in wild-type cells presented an average replication speed of 1.11 kb/min, the ones in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells had their processivity almost cut in half (0.72 kb/min and 0.53 kb/min for HeLa 2.7 and HeLa 4.3 respectively). Since replication of the leading and lagging strands involves different DNA polymerases, each with their own replication speed\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, the symmetry of elongating fork was also measured by dividing the length of the longer branch (if any) and divided by the shorter branch (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-g). Only replication forks featuring the origin site and the successive CldU and IdU labelling were considered in this analysis. Overall, Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells presented a slight increase of fork asymmetry. This variation, however, was found significant only for one of the two clones: HeLa 4.3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Ultimately, all these observations showed that PCNA modification by Ub\u003csup\u003eKEKS\u003c/sup\u003e plays a role (direct or indirect) in the regulation of DNA polymerases processivity. However, whether Ub\u003csup\u003eKEKS\u003c/sup\u003e also contribute to the discrimination between leading and lagging strands remains to be confirmed.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePCNA forms an homotrimer that binds to DNA and acts as a recruitment platform for proteins involved in various cellular processes, including origin firing\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, increasing DNA polymerases processivity\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, Okazaki fragments maturation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, DNA damage tolerance\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and DNA repair\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The regulation of these pathways heavily relies on the competitive binding affinity between unmodified or post-translationally modified PCNA and its interacting partners\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. PTMs such as Ub and Ub-like proteins play a central role in fine-tuning those interactions by introducing additional binding sites via recognition sequences\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Ub\u003csup\u003eKEKS\u003c/sup\u003e is a recently discovered PTM that can specifically modify proteins and has been implicated in nucleolar composition and apoptosis regulation\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Here, we demonstrate that PCNA is one of the major targets of Ub\u003csup\u003eKEKS\u003c/sup\u003e. Our findings identified lysine 164 as the primary modification site, a residue widely recognized as a critical molecular switch for PCNA-mediated pathway regulation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e modifications do not compensate for each other, we showed that K164 can be modified by either of them. These two modifications likely rely on competitive binding as over-expression of Ub\u003csup\u003eKEKS\u003c/sup\u003e shifted the ratio of Ub\u003csup\u003eKEKS\u003c/sup\u003e and Ub covalently bound to PCNA. To explore the functional consequences of this modification, we used Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells, which exhibit a proliferation delay.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Given PCNA\u0026rsquo;s crucial role in DNA replication\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we investigated S-phase progression in these cells. Our results revealed that Ub\u003csup\u003eKEKS\u003c/sup\u003e deficiency induces a significant S-phase delay without affecting the overall cell cycle distribution. This delay was not attributable to alterations in PCNA abundance or its subnuclear localization. Instead, we observed a marked reduction in DNA polymerases processivity, with replication fork speed halved in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells. These findings suggest an essential role for Ub\u003csup\u003eKEKS\u003c/sup\u003e in regulating replication fork dynamics.\u003c/p\u003e \u003cp\u003ePCNA can be subject to multiple PTMs, including phosphorylation, acetylation, methylation or ubiquitination\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31 CR32 CR33 CR34\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Using affinity purification via the AviTag system and co-immunoprecipitation against GFP-tagged proteins, we established that Ub\u003csup\u003eKEKS\u003c/sup\u003e is covalently attached to PCNA, adding a novel layer of regulation. Notably, Ub\u003csup\u003eKEKS\u003c/sup\u003e does not modify PCNA with the same preferences as compared with Ub, targeting mainly K164. This residue is essential for protein recruitment and serves as a molecular switch coordinating PCNA-mediated mechanisms\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Additionally, we detected simultaneous modifications of PCNA by two PTMs, with at least one being Ub\u003csup\u003eKEKS\u003c/sup\u003e. However, it remains unclear whether this dual modification involves two Ub\u003csup\u003eKEKS\u003c/sup\u003e monomers on different lysines, a combination of Ub\u003csup\u003eKEKS\u003c/sup\u003e and Ub, or whether it forms a polychain.\u003c/p\u003e \u003cp\u003eRegarding the functional implications of Ub\u003csup\u003eKEKS\u003c/sup\u003e modification, multiple possibilities exist due to PCNA\u0026rsquo;s involvement in numerous cellular pathways\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Since PCNA plays a central role in DNA replication and repair, and Ub\u003csup\u003eKEKS\u003c/sup\u003e deficiency leads to a proliferation delay\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, we focused on S-phase regulation. The delay was not linked to changes in PCNA abundance, aligning with previous findings that Ub\u003csup\u003eKEKS\u003c/sup\u003e does not target proteins for proteasomal degradation\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Furthermore, despite Ub\u003csup\u003eKEKS\u003c/sup\u003e influencing the subcellular localization of nuclear and nucleolar proteins\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, PCNA localization during S-phase remained unaffected in Ub\u003csup\u003eKEKS\u003c/sup\u003e KO cells, suggesting that impaired PCNA recruitment to the replication fork does not account for the observed delay. Instead, our data indicate that Ub\u003csup\u003eKEKS\u003c/sup\u003e depletion significantly reduces DNA polymerase processivity. Normally, PCNA recruits and stabilizes DNA polymerases, enhancing their processivity by up to 100-fold\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. PCNA interactors compete for recruitment, influenced by PCNA-binding motifs, PTM modifications, and proximity to specific factors\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Given this, Ub\u003csup\u003eKEKS\u003c/sup\u003e likely serves as a crucial new regulator of DNA polymerase recruitment and stabilization by functioning as a PTM and providing additional recognition sequences. Moreover, Ub\u003csup\u003eKEKS\u003c/sup\u003e depletion correlated with an increase in asymmetric replication forks. Proximity to regulatory proteins such as the CMG complex or RPA proteins, is known to differentiate leading from lagging strand synthesis, facilitating selective recruitment of DNA polymerases\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This raises the possibility that Ub\u003csup\u003eKEKS\u003c/sup\u003e plays a role in distinguishing DNA strands, either by modifying PCNA itself or by acting on another regulatory factor. Collectively, these findings highlight Ub\u003csup\u003eKEKS\u003c/sup\u003e as a key regulator of DNA replication.\u003c/p\u003e \u003cp\u003eOn a broader scale, our findings illustrate the emergence of an entirely new research avenue concerning ubiquitylation. Both Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e specifically target a wide array of proteins\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Analysis of PCNA modification sites and immunoprecipitation against diGly signatures confirmed that Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e target different residues and do not compensate for each other at the cellular level. Although no strong consensus sequence for Ub\u003csup\u003eKEKS\u003c/sup\u003e modification emerged, we nevertheless observed an enrichment of lysines and methionines around its target sites, which contrast with the lack of lysines, methionines, and cysteines around residues modified by Ub\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. This inverse correlation in residue composition may be a distinguishing feature between Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e target sites. Additionally, the selection of modification sites is largely determined by steric constraints imposed by E2 and E3 enzymes involved in the modification cascade\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Like Ub and other Ub-like proteins, Ub\u003csup\u003eKEKS\u003c/sup\u003e likely employs an enzymatic conjugation system, as suggested by the significant enrichment of E1 enzyme UBA1 and E2 enzyme UBE2N/UBE2NL in our AviTag pulldowns. This Ub\u003csup\u003eKEKS\u003c/sup\u003e enzymatic cascade, however, potentially involve distinct combinations E2 and E3 enzymes.\u003c/p\u003e \u003cp\u003eOverall, our findings define Ub\u003csup\u003eKEKS\u003c/sup\u003e as a novel post-translational regulator of PCNA, adding a new dimension to its already intricate PTM-mediated regulation. Ub\u003csup\u003eKEKS\u003c/sup\u003e plays a critical role in DNA replication by enhancing DNA polymerase processivity. More broadly, Ub\u003csup\u003eKEKS\u003c/sup\u003e emerges as a unique PTM, distinct from Ub, targeting different proteins, and potentially utilizing a separate enzymatic conjugation pathway. These insights pave the way for further exploration of Ub\u003csup\u003eKEKS\u003c/sup\u003e as a universal PTM and its implications in cellular regulation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHeLa cells were cultured under normal conditions (37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e), as adherent cells in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% FB Essence and 100U/mL Penicillin/streptomycin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification of Ub targets with AviTag constructs\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eGeneration of plasmids.\u003c/em\u003e AviTag constructs were designed to simultaneously express several tagged proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea \u0026ndash; Supp Table\u0026nbsp;1). Plasmids creation required two main steps: i) generation of the basic construction called pDONR-AviTag and ii) insertion of the sequences of the protein of interest (either tag HA alone, HA-tagged non-cleavable Ub\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e or HA-tagged Ub\u003csup\u003eKEKS\u003c/sup\u003e). The non-cleavable version of Ub was used to maximize \u003cem\u003ein-cellulo\u003c/em\u003e labelling of ubiquitinated proteins\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. All sequences from the gene fragments used to create our plasmids are available in Supp Table\u0026nbsp;1. To create the pDONR-AviTag plasmid, a 1181 bp DNA gene fragment (Integrated DNA technologies, optimized sequence for mammalian cell expression) was designed to contain the following elements (Supp Table\u0026nbsp;1): an attb1 site followed by a KOZAK consensus sequence, the AviTag peptide sequence (GLNDIFEAQKIEWHE\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e), a unique \u003cem\u003eKpn\u003c/em\u003eI restriction site, \u003cem\u003eE. coli\u003c/em\u003e\u0026rsquo;s BirA\u003csup\u003ewt\u003c/sup\u003e protein sequence with a C-terminal myc-tag sequence, a Glycine-Serine-Glycine (GSG) linker followed by a T2A self-cleaving peptide sequence\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and, finally, an attb2 site. This gene fragment was used in a BP recombination reaction using BP Clonase II (Invitrogen #11789020) in combination with the \u003cem\u003eattP\u003c/em\u003e-containing donor vector pDONR221 (Invitrogen #12536017) to generate the pDONR-AviTag plasmid. Next, three different gene fragments were ordered from Integrated DNA technologies as future inserts (Supp Table\u0026nbsp;1). These gene fragments contained a Gibson assembly 5\u0026rsquo;-end pairing sequence, a \u003cem\u003eKpn\u003c/em\u003eI restriction site, the HA tag sequence either alone, fused to Ub or fused to Ub\u003csup\u003eKEKS\u003c/sup\u003e DNA sequence, a GSG linker by a T2A self-cleaving peptide sequence as described before, a second \u003cem\u003eKpn\u003c/em\u003eI restriction site and, finally, a Gibson assembly 3\u0026rsquo;-end pairing sequence. Each gene fragment was used in a Gibson assembly reaction (New England Biolabs #M5510A) with the \u003cem\u003eKpn\u003c/em\u003eI-digested pDONR-AviTag plasmid. Resulting \u003cem\u003eattL\u003c/em\u003e-containing pDONR constructs were then used in LR recombination reaction using LR Clonase II (Invitrogen #11791100) with either the \u003cem\u003eattR\u003c/em\u003e-containing destination vector pDEST47 (Invitrogen #112281010) for HA tag alone and HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e; or the \u003cem\u003eattR\u003c/em\u003e-containing destination vector pGLAP5.2 (gift from Peter Jackson, Addgene plasmid #19706). Obtained fusion proteins are summarized up in Supp Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBiotin labelling and pulldown assay.\u003c/em\u003e Wild-type HeLa cells were transfected with either the control-AviTag, the Ub-AviTag or the Ub\u003csup\u003eKEKS\u003c/sup\u003e-AviTag plasmids (Supp Table\u0026nbsp;1). Cells were cultivated under normal conditions for 48h, then incubated with biotin for another 24h. Next, cells were washed with PBS 1X and scrapped in denaturing lysis buffer (8 M urea, 20 mM HEPES, 10 mM DTT). Samples were sonicated on ice for 1 min at 30% (Fischer Scientific Model 120 Sonic Dismembrator) with 5s on-5s off cycles. The lysates were then centrifuged at 4\u0026deg;C for 10 min at 16 000g to remove cellular debris. For each sample, 1 mg of total protein extract was incubated for 2h at room temperature with 25\u0026micro;l of high-performance streptavidin beads (Cytiva #17511301). Beads were then washed four times with lysis buffer before being transferred into low-bind tubes after the final wash. Finally, samples were prepared following the on-bead digestion protocol described below and send for Data Independent Acquisition (DIA) mass spectrometry.\u003c/p\u003e\n\u003ch3\u003eConfirmation of PCNA modification by Ub\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eCloning of the PCNA-GFP protein\u003c/em\u003e. mRNA was extracted from exponentially growing wild-type HeLa cells using TRIzol reagent (Invitrogen #15596026), according to the manufacturer\u0026rsquo;s instructions. PCNA sequence was amplified by RT-PCR with the primers PCNA_attb1 and PCNA_attb2, available in Supp Table\u0026nbsp;3. These oligos allowed to insert ATTB sites for Gateway cloning. A first recombination was performed from the PCR product into the pDONR221 plasmid (Invitrogen #12536017) using the BP Clonase II (Invitrogen #11789020), following the manufacturer\u0026rsquo;s protocol. Resulting plasmids were transformed into thermo-competent bacteria which were then seeded on LB plate with kanamycin and grown overnight. On the next morning, isolated colonies were harvested: plasmid constructions were extracted by mini-prep and validated by sequencing. Correct plasmids went under a LR recombination step into the pDEST47 plasmid (Invitrogen #112281010) using the LR Clonase II (Invitrogen #11791100). Plasmid final validations were done by sequencing.\u003c/p\u003e \u003cp\u003e \u003cem\u003eProtein overexpression and immunoprecipitation.\u003c/em\u003e Wild-type HeLa cells were cultivated in 6-wells plates until 60\u0026ndash;70% confluency was reached. PCNA-GFP plasmid in combination with either the empty plasmid (pcDNA 3.1), HA-Ub or HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e plasmid were co-transfected using Lipofectamine 2000 (Invitrogen #11668019), following the manufacturer\u0026rsquo;s guidelines and incubated for 48h. Cells were washed with PBS 1X and lysed in High Salt buffer (50 mM Tris pH 7.5, 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 1 mM DTT, 10% Glycerol, 1% NP40, PMSF 1X). Samples were sonicated on ice for 10s at 25% (Fischer Scientific Model 120 Sonic Dismembrator). The lysates were then centrifuged at 4\u0026deg;C for 10 min at 12 000g to remove cellular debris. 1 mg of each sample was incubated overnight at 4\u0026deg;C, with GFP-Trap agarose beads (ChromoTek, Martinsried, Germany). Beads were washed three times with High Salt buffer, before being incubated in Laemmli and boiled at 95\u0026deg;C for 5 min, to elute proteins. Samples were resolved on SDS-PAGE and transferred onto nitrocellulose membrane. Signals were revealed using GFP (Roche #11814460001, dilution 1:1000) and HA (Invitrogen #26183, dilution 1:1000) antibodies.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of modification sites on PCNA\u003c/h2\u003e \u003cp\u003e \u003cem\u003eChoice of lysine according to pre-existing databases.\u003c/em\u003e Lysines potentially modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e were selected based on the Ub modification sites listed in the mUbiSiDa website, a mammalian Ubiquitination Site Database \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Database was last accessed in April 2019. Eight lysines were chosen for mutated forms of PCNA: K13, K14, K77, K80, K110, K164, K248 and K254.\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuadruple-mutated PCNA plasmids creation using gene fragments.\u003c/em\u003e Gene fragments containing the desired 4 mutations with attb1 and attb2 sites at each extremity were ordered from Integrated DNA Technologies (sequences available in Supp Table\u0026nbsp;3). As described in the previous section, final plasmids were generated using Gateway cloning technology. Gene fragments were mixed with pDONR221 plasmid (Invitrogen #12536017) and BP Clonase II (Invitrogen #11789020), and incubated at 25\u0026deg;C according to the manufacturer\u0026rsquo;s protocol. After transformation into thermo-competent cells and validation of the extracted plasmids by sequencing, correct products were incubated with pDEST47 plasmid (Invitrogen #112281010) and LR Clonase II (Invitrogen #11791100) following the manufacturer\u0026rsquo;s guidelines. Plasmid final validations were done by sequencing.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGeneration of single-mutation PCNA plasmid by site-directed mutagenesis.\u003c/em\u003e Plasmids containing mutated forms of PCNA were generated from the plasmid expressing wild-type PCNA-GFP, using site-directed mutagenic PCR as described by Liu et Naismith\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Briefly, 50 ng of DNA template was amplified by iProof DNA polymerase (BioRad #1725301), using both forward and reverse primers containing the desired mutation. Obtained PCR products were digested by DpnI for 1h, and the plasmids were transformed in DH10B thermocompetent \u003cem\u003eE. coli\u003c/em\u003e bacteria (Thermo Scientific #EC0113). Plasmids were purified using a mini-prep kit (Favorgen Biotech Corp. #FAPDE-100) and final products were validated by enzymatic digestion and sequencing. Primers used to generate PCNA mutated plasmids are available in Supp Table\u0026nbsp;3.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOverexpression and immunoprecipitation of mutated forms of PCNA.\u003c/em\u003e As described for the previous experiment, wild-type HeLa cells were cultured in 6-well plates until reaching 60\u0026ndash;70% confluency. HA-Ub or HA-Ub\u003csup\u003eKEKS\u003c/sup\u003e plasmid along with a plasmid coding for different mutated forms of PCNA were co-transfected using Lipofectamine 2000 (Invitrogen #11668019). After 48h, cells were washed with PBS 1X and lysed in High Salt buffer (50 mM Tris pH 7.5, 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 1 mM DTT, 10% Glycerol, 1% NP40, PMSF 1X). Samples were sonicated on ice for 10s at 25% (Fischer Scientific Model 120 Sonic Dismembrator) and then centrifuged at 4\u0026deg;C for 10 min at 12 000g to remove cellular debris. 1 mg of each sample was incubated overnight at 4\u0026deg;C, with GFP-Trap agarose beads (ChromoTek, Martinsried, Germany). Beads were washed three times with High Salt buffer. Beads were then incubated in Laemmli and boiled at 95\u0026deg;C for 5 min, to elute proteins. Samples were resolved on SDS-PAGE and transferred onto nitrocellulose membrane. Loss of modification by Ub or by Ub\u003csup\u003eKEKS\u003c/sup\u003e was detected using GFP (Roche #11814460001, dilution 1:1000) and HA (Invitrogen #26183, dilution 1:1000) antibodies.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUb Knock-out HeLa cells\u003c/h3\u003e\n\u003cp\u003eHeLa cells invalidated for Ub\u003csup\u003eKEKS\u003c/sup\u003e were previously generated using the CRISPR/Cas9 method\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In this article, \u0026ldquo;HeLa 2.7\u0026rdquo; and \u0026ldquo;HeLa 4.3\u0026rdquo; refer to Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells using two different combinations of sgRNAs to invalidate the UBBP4 pseudogene. Unless specified otherwise, these cells were also cultured as adherent cells and in the same conditions as those described in the previous paragraph on cell culture.\u003c/p\u003e\n\u003ch3\u003eCharacterization of Ub and Ub modifications on PCNA by mass spectrometry\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eSILAC labelling.\u003c/em\u003e Wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells were cultured in DMEM depleted of arginine and lysine (Life Technologies #A14431) which was supplemented with 10% triple dialyzed fetal bovine serum (Invitrogen #26400-044), 100U/mL Penicillin/streptomycin and 2 mM of GlutaMax (Gibco #35050-061). Lysines and arginines were added to create either light (Arg\u003csub\u003e0\u003c/sub\u003eLys\u003csub\u003e0\u003c/sub\u003e, Sigma-Aldrich #A5006 and Sigma-Aldrich #L5501) or medium (Arg\u003csub\u003e6\u003c/sub\u003eLys\u003csub\u003e4\u003c/sub\u003e, Cambridge Isotope Lab #CNM-2265 and Cambridge Isotope Lab # DLM-2640) labelled media. Final concentrations of each type of medium were 28\u0026micro;g/mL for arginine and 49\u0026micro;g/mL for lysine. L-proline was also added to reach a final concentration of 10 \u0026micro;g/mL and therefore, prevent unwanted arginine to proline conversion. Wild-type cells were labelled with light SILAC whereas knockout clones 2.7 and 4.3 were labelled in medium SILAC.\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunoprecipitation and preparation for mass spectrometry.\u003c/em\u003e Seven 150 mm plates of wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells were grown SILAC medium until confluency to obtain 20 mg of total cell extract. Proteins from each SILAC medium were mixed together to create two distinct solutions: either HeLa WT with HeLa 2.7 or HeLa WT with HeLa 4.3 (both solutions contained 1:1 ratio of SILAC labelled total extracts). Detection of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e modification sites was made using the PTMScan\u0026reg; Pilot Ubiquitin Remnant Motif K-ε-GG kit (Cell Signaling Technology #14482)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea): cell lysis, protein digestion, purification of lysate peptides and immunoaffinity purification of modified peptides were performed according to the manufacturer\u0026rsquo;s protocol. Final peptides obtained after all those steps were desalted and concentrated using ZipTips, prior being sent to the mass spectrometer for data-dependent acquisition (DDA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the environment of Ub\u003csup\u003eKEKS\u003c/sup\u003e-modified lysines\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePython programming.\u003c/em\u003e Two short Python scripts were written to determine the number of appearances for each amino acid surrounding lysines of interest. The first script is designed for datasets containing truncated protein sequences measuring exactly 31 residues, with the lysine of interest at position 16. This type of data can typically be obtained using the diGly analysis featured in the MaxQuant\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e software for mass spectrometry experiments. The second script can be used on any dataset containing full protein sequences under a FASTA format. Both scripts rely on a string-based analysis and result in a final matrix indicating the number of appearances for each residue depending on their position within the lysine\u0026rsquo;s environment. Briefly, these scripts read the environment of each lysine given to the program by the user and identify the type of amino acid in each position. When a specific residue is identified within the surrounding of a lysine of interest, its associated counter in the final matrix is incremented. Once all lysines imputed by the user have been analyzed, the script returns the final matrix. Both Python scripts were made public on the GitHub repository at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/fmboisvert/KAnalysis\u003c/span\u003e\u003cspan address=\"https://github.com/fmboisvert/KAnalysis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, under the Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) license. For additional information and a short user manual, please refer directly to the files uploaded on GitHub.\u003c/p\u003e \u003cp\u003e \u003cem\u003eApplying the program to biological data.\u003c/em\u003e Python scripts described above were tested on 2 distinct datasets. On one hand, a list containing the residues sequences surrounding 655 lysines specifically modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e were imputed into the first script. This list was obtained by selecting lysines presenting a significant drop of diGly motifs in Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells (please refer to the paragraph entitled \u0026ldquo;Mass spectrometry data analysis\u0026rdquo; below). On the other hand, the Uniprot public database (20 386 entries, last accessed on October 27, 2021) was imputed into the second Python script to evaluate the average environment composition surrounding lysine in humans. This second analysis was defined as a control condition to compare the output of both scripts and therefore identify a potential enrichment or depletion of specific residues around modification sites specific to Ub\u003csup\u003eKEKS\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of the Ub\u003csup\u003eKEKS\u003c/sup\u003e:Ub ratio on PCNA by mass spectrometry\u003c/h2\u003e \u003cp\u003eWild-type HeLa cells were cultured in 100 mm dishes under normal conditions until ~\u0026thinsp;70% confluency. Cells were the co-transfected with a plasmid expressing PCNA-GFP along either a control plasmid (empty pcDNA3.1), a plasmid expressing Ub-HA or a plasmid expressing Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA. Co-transfections were done using Lipofectamine 2000, according to the manufacturer\u0026rsquo;s instructions (Invitrogen #11668019). After 48h, cells were lysed in High Salt Buffer (50 mM Tris pH 7.5, 300 mM NaCl, 150 mM KCl, 5 mM EDTA, 1 mM DTT, 10% Glycerol, 1% NP40, PMSF 1X, Roche Complete Protease Inhibitor Cocktail) and sonicated on ice for 10s at 35% (Fischer Scientific Model 120 Sonic Dismembrator). Lysates were then centrifuged at 4\u0026deg;C for 10 min at 12 000g. 1 mg of each sample was incubated with GFP-Trap agarose beads (ChromoTek, Martinsried, Germany) at 4\u0026deg;C, overnight. Beads were then washed 3 times with high-salt buffer and proteins were eluted using 2x Laemmli containing 10 mM DTT. Beads were boiled at 95\u0026deg;C for 5 min and let at room temperature to cooldown. Chloroacetamide (Sigma Aldrich #22790) was added to reach a final concentration of 50 mM and beads were incubated in the dark for 30 min. Following alkylation, samples were separated on a 4\u0026ndash;12% gradient SDS-PAGE gel. Gel was the stained using SimplyBlue SafeStain (Invitrogen) and destained overnight in distilled water. Gel regions containing modified and unmodified PCNA-GFP were excised and proteins were digested in gel overnight using trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega Corporation, WI, USA). Peptides were then extracted from the gel pieces and a mix of unique heavy peptides were spiked into the samples at a final concentration of 1.66fmol/\u0026micro;l. This mix is composed of 4 heavy peptides with a 1:1:1:1 ratio: AQUA Ub (U-13C6, U-15N4; mass difference: +10 Da; sequence EGIPPDQQR \u0026ndash; ThermoFischer Scientific), AQUA Ub\u003csup\u003eKEKS\u003c/sup\u003e (U-13C6, U-15N4; mass difference: +10 Da; sequence IQDEEGIPPDQQR - ThermoFischer Scientific), NEP Ub (U-13C6, U-15N; U-13C6, U-15N2; mass difference: +15 Da; sequence TLSDYNIQK - New England Peptide), NEP Ub\u003csup\u003eKEKS\u003c/sup\u003e (U-13C6, U-15N; U-13C6, U-15N2; mass difference: +15 Da; sequence TLSDYSIQK - New England Peptide). Then, the peptides were desalted using ZipTips C18 columns (EMD Millipore, Burlington, VT). Samples were dried by speed vac and resuspend in 1% formic acid. Peptides were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) at a wavelength of 205 nm. The peptides were then transferred into a glass vial (Thermo Fisher Scientific) and kept at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis by mass spectrometry using PRM quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStudy of the cell cycle of wild-type and knockout cells\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS phase progression monitoring.\u003c/em\u003e 200 000 wild-type and knockout cells were cultured in 6-wells plates under normal conditions until reaching 80% of confluency. Cells were labelled with warm medium containing 10\u0026micro;g of BrdU for 1h at 37\u0026deg;C, and then washed with warm PBS 1X, before warm medium without BrdU was added (BrdU chase). At 0h, 4h and 8h after the change of medium, cells were fixed in ethanol 70% at -20\u0026deg;C for at least 48h. Fixed cells were centrifuged and resuspended in a 2 N HCl and 0.5% of Triton X-100 solution. Samples were then incubated for 30 min at room temperature before being recentrifuged once again. Cell pellets were gently resuspended in NaB\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e 0.1M (pH 8.5) and incubated for another 30 min at room temperature. Cells were centrifuged and pellets were resuspended in antibody solution (1% BSA, 0.2% Tween-20 in PBS 1X) containing mouse BrdU antibodies (BD Biosciences #347580, dilution 1:50). After a 30 min incubation at room temperature, cells were centrifuged and resuspended in antibody solution with anti-mouse Alexa Fluor 488 antibodies (Invitrogen, #A-11001, dilution 1:50), for 30 min at room temperature in the dark. Cells were centrifuged one last time and finally incubated in the dark for 30 min in a propidium iodide-RNAse solution (10 mg/mL propidium iodide, 0.25 mg/mL RNAse A in PBS 1X). Fluorophores signals were acquired by flow cytometry (BD Fortessa cytometer, Becton Dickinson) and analyzed with FlowJo, version 10.8.1 (Becton Dickinson \u0026amp; company). BrdU signal was plotted against propidium iodide labelling to precisely detect all cell cycle phases. Percentages of cells in late S phase at 4h and in new G1 phase at 8h were measured. For both time points, percentages of cells for each of the populations went under statistical analysis in GraphPad (GraphPad Software - version 9.0.0., USA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell cycle distribution analysis.\u003c/em\u003e The samples prepared for S-phase progression monitoring were also used to determine the cell cycle distribution in wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells. Percentages of cells in G1, S and G2/M phases went under statistical analysis in GraphPad (GraphPad Software - version 9.0.0., USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAbundance of intracellular PCNA\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAnalysis of mass spectrometry data comparing the total proteomes of wild-type and knockout cells.\u003c/em\u003e Mass spectrometry data generated by Data Independent Acquisition (DIA)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and available on the ProteomeXchange Consortium via the PRIDE\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e partner repository under the dataset identifier PXD040778; were analyzed by comparing whole cell proteomes from wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells. Data were loaded into the ProStaR software\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e (version 1.26.1). Identified proteins with a unique peptide count lower than 2 were filtered out and partially observed missing values were imputed with the slsa algorithm. Missing values for entire conditions were imputed using the Det quantile algorithm (Quantile\u0026thinsp;=\u0026thinsp;1; Factor\u0026thinsp;=\u0026thinsp;0,2). A differential abundance analysis was later performed in ProStaR to evaluate the impact of Ub\u003csup\u003eKEKS\u003c/sup\u003e on PCNA\u0026rsquo;s cellular levels. Comparison was made using the following parameters: sequence coverage, the number of unique peptides identified and the intensity detected for PCNA.\u003c/p\u003e \u003cp\u003e \u003cem\u003eConfirmation by western blot.\u003c/em\u003e Exponentially growing wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells were lysed in clear Laemmli buffer and sonicated on ice (Fischer Scientific Model 120 Sonic Dismembrator). Samples were centrifuged at 12 000g for 10 min (4\u0026deg;C) to get rid of cellular debris and 25\u0026micro;g of the supernatants were resolved by SDS-PAGE prior transfer on nitrocellulose membrane. Signals were detected with endogenous PCNA antibodies (Cell Signaling #2586S, dilution 1:1000) while \u0026szlig;-tubulin antibodies (Cell Signaling #2128S, dilution 1:1000) were used as loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNuclear localization of PCNA during S-phase\u003c/h2\u003e \u003cp\u003eWild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout cells were seeded on glass coverslips in a 24-wells plate until 40% confluency. Cells were washed in PBS 1X and fixed for 15 min with 4% formaldehyde in PBS 1X at room temperature. Coverslips were washed twice with PBS 1X and cells were permeabilized for 10 min at -20\u0026deg;C, using ice-cold 100% methanol. Cells were washed again with PBS 1X prior to incubation for 1h in blocking buffer (5% Goat serum, 0.3% Triton X-100 in PBS 1X). Endogenous PCNA was labelled by using PCNA antibodies (Cell Signaling #2586S, dilution 1:2000) in blocking buffer (overnight incubation, 4\u0026deg;C). Cells were then washed three times with PBS 1X and incubated in blocking buffer containing goat anti-mouse Alexa Fluor 488 antibodies (Invitrogen, #A-11001, dilution 1:800) for 1h in the dark, at room temperature. Nuclei were stained with DAPI solution (1\u0026micro;g/\u0026micro;l) for 8 min in PBS 1X and cells were washed again twice. Coverslips were mounted on microscope slides using Immuno-mount medium (ThermoFischer Scientific). Images were acquired on a Zeiss LSM 880 confocal microscope using a 40\u0026times; 1.4NA plan Apo objective. Image treatment and assembling were performed using Fiji (version 1.53c) software\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Image classification was based on peer-reviewed literature\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Nuclear locations of PCNA during S-phase are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec while PCNA\u0026rsquo;s locations during other cell cycle phases are available in Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eReplication combing assay\u003c/h2\u003e \u003cp\u003eExponentially growing wild-type and Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout HeLa cells were cultured in a 6-wells plate until 80% confluency. Cells were labelled 45 min with 100\u0026micro;M of 5-chloro-2\u0026rsquo;-deoxyuridine (CldU, Sigma-Aldrich #C6891), followed by another incubation of 45 min with 100\u0026micro;M of 5-iodo-2\u0026rsquo;-deoxyuridine (IdU, Sigma-Aldrich #I7125). Cells were trypsinized and counted before being embedded into agarose plugs and going under DNA extraction following the protocol described in the FiberPrep kit (Genomic Vision, Bagneux, France). Coverslips coated with vinylsilane were combed with a constant 2 kb/\u0026micro;m stretching factor, using the molecular combing system FiberComb (Genomic Vision, Bagneux, France). Coverslips were baked for 2h at 60\u0026deg;C in the dark, then denatured and blocked with BlockAid solution (Invitrogen #B10710). DNA strands were labelled using mouse ssDNA (DSHB University of Iowa, United States), mouse BrdU (favors IdU labelling, BD Biosciences #347580), rat BrdU (favors CldU labelling, Abcam #AB6326) antibodies. After three PBS-Tween washes, secondary antibodies staining was performed with goat anti-mouse BV480 (BD Biosciences #564877), goat anti-mouse Alexa Fluor 555 (Invitrogen #A21424) and goat anti-rat Cy5 (Abcam, #AB6565). All antibodies dilutions were done following Genomic Vision\u0026rsquo;s protocol for replication combing assay. Coverslips were dehydrated by ethanol, mounted and pictures were acquired using the EasyScan service (Genomic Vision, Bagneux, France). DNA fibers were analyzed using the FiberStudio software. Fork speed for each cell line was calculated using the formula \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:V\\:=\\:\\left(fiber\\:length\\times\\:\\:stretching\\:factor\\right)\u0026divide;labelling\\:time\\)\u003c/span\u003e\u003c/span\u003e. All statistics were performed using Graph Pad Prism version 9.0.0. (GraphPad Software, USA). To facilitate the observation of elongation forks, only signals corresponding to incorporated CldU (red) and incorporated IdU (green) are shown in this article.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOn-beads digestion of samples for mass spectrometry\u003c/h2\u003e \u003cp\u003eBeads bound to proteins of interest were washed five times with 20 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e. After the final wash, beads were resuspended in 50\u0026micro;l of 10 mM DTT and 20 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e, and incubated at 60\u0026deg;C for 30 min under agitation. Then, beads were left to cooldown before adding 50\u0026micro;l of 15 mM chloroacetamide (Sigma Aldrich #22790) in 20 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e. Samples were incubated 1h in the dark at room temperature. DTT concentration in samples was raised to 15 mM to quench chloroacetamide. After a 10 min incubation at room temperature, 1\u0026micro;g of trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega Corporation, WI, USA) was added to the beads and tubes were incubated at 37\u0026deg;C overnight. Digestion was then stopped by acidifying the beads to a final concentration of 1% formic acid. Supernatants were collected and transferred into new lowbind tubes. Beads were resuspended in 60% acetonitrile and 1% formic acid solution before a 5 min incubation at room temperature. Supernatants were once again harvested and mix with their corresponding supernatants previously collected. Samples were dried by speedvac and desalted using ZipTips C18 column (EMD Millipore, Burlington, VT), and finally resuspended in 1% formic acid. Peptides were quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) at a wavelength of 205 nm. The peptides were then transferred into a glass vial (Thermo Fisher Scientific) and kept at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis by mass spectrometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry acquisition\u003c/h2\u003e \u003cp\u003e \u003cem\u003eData-independent acquisition (DIA).\u003c/em\u003e This method of acquisition was used to generate results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. 250 ng of peptides from each sample were injected into an HPLC (nanoElute, Bruker Daltonics) and loaded onto a trap column with a constant flow of 12 \u0026micro;l/min (Acclaim PepMap100 C18 column, 0.3 mm id x 5 mm, Dionex Corporation); then eluted onto an analytical C18 Column heated to 50\u0026deg;C (1.9 \u0026micro;m beads size, 75 \u0026micro;m x 25 cm, PepSep). Peptides were eluted over a 2-hour gradient of ACN (5\u0026ndash;37%) in 0.1% FA at 400 nL/min while being injected into a TimsTOF Pro ion mobility mass spectrometer equipped with a Captive Spray nano electrospray source (Bruker Daltonics). Data were acquired using diaPASEF with a 100\u0026ndash;1700 m/z mass range for TIMS-MS scan. For each single TIMS (100 ms) in diaPASEF mode, we used 1 mobility window consisting of 27 mass steps (m/z between 114 to 1414 with a mass width of 50 Da) per cycle (1.27 seconds duty cycle). These steps cover the diagonal scan line for +\u0026thinsp;2 and +\u0026thinsp;3 charged peptides in the m/z-ion mobility plane. Raw files were analyzed using DIA-NN\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e and the Uniprot human proteome database (version : March 21, 2020\u0026ndash;75 776 entries). Further details are available in the Mass spectrometry data analysis paragraph.\u003c/p\u003e \u003cp\u003e \u003cem\u003eData-dependent acquisition (DDA).\u003c/em\u003e This method of acquisition was used to generate results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec. Trypsin-digested peptides were loaded and separated onto a nanoHPLC system (Dionex Ultimate 3000). A total of 10 \u0026micro;l of the sample (1.5 \u0026micro;g) was first loaded with a constant flow of 4 \u0026micro;l/min onto a trap column (Acclaim PepMap100 C18 column, 0.3 mm id \u0026times; 5 mm, Dionex Corporation). Peptides were then eluted off towards an analytical C18 column heated to 40\u0026deg;C (2 \u0026micro;m beads size, 75 \u0026micro;m \u0026times; 50 cm, PepMap) with a linear gradient of 5\u0026ndash;45% of solvent B (80% acetonitrile with 0.1% formic acid) over a 4 h gradient at a constant flow (200 nL/min). Peptides were then analyzed by an Orbitrap Q Exactive mass spectrometer (Thermo Scientific) using an EasySpray source at a voltage of 2.0 kV. Acquisition of the full scan MS survey spectra (m/z 350\u0026ndash;1600) in profile mode was performed in the Orbitrap at a resolution of 70,000 using 1,000,000 ions. Peptides selected for fragmentation by collision-induced dissociation were based on the ten highest intensities for the peptide ions from the MS survey scan. The collision energy was set at 25% and resolution for the MS/MS was set at 17,500 for 500,000 ions with maximum filling times of 20 ms for the full scans and 60 ms for the MS/MS scans. All unassigned charge states as well as singly, seven and eight charged species for the precursor ions were rejected, and a dynamic exclusion list was set to 500 entries with a retention time of 40 s (10 ppm mass window). To improve the mass accuracy of survey scans, the lock mass option was enabled. Data acquisition was done using Xcalibur version 4.3.73.11. Identification and quantification of proteins identified by MS were done using the MaxQuant software version 1.5.2.8.\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Biological replicates were done three times and combined together for the MaxQuant analysis.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePRM quantification of Ub\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e:\u003cem\u003eUb modification of PCNA.\u003c/em\u003e This method of acquisition was used to generate results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Peptides were loaded and separated onto a nanoHPLC system (Dionex Ultimate 3000) with a constant flow of 4 \u0026micro;l/min onto a trap column (Acclaim PepMap100 C18 column, 0.3 mm id \u0026times; 5 mm, Dionex Corporation, Sunnyvale, CA). Peptides were then eluted off towards an analytical C18 column heated to 40\u0026deg;C (2 \u0026micro;m beads size, 75 \u0026micro;m \u0026times; 50 cm, PepMap) with a linear gradient of 5\u0026ndash;45% of solvent B (80% acetonitrile with 0.1% formic acid) over a 42-min gradient at a constant flow (400 nL/min). Peptides were analyzed by an Orbitrap Q Exactive mass spectrometer (Thermo Scientific) using a Parallel Reaction Monitoring (PRM) method. An inclusion list containing the m/z values corresponding to the monoisotopic form of the heavy and light peptides of Ub (AQUA Ub :520.3/525.3 ; NEP Ub: 541.3/548.8) and Ub\u003csup\u003eKEKS\u003c/sup\u003e (AQUA Ub\u003csup\u003eKEKS\u003c/sup\u003e: 762.9/767.9 ; NEP Ub\u003csup\u003eKEKS\u003c/sup\u003e: 527.8/535.3) was generated. Acquisition of the MS spectra (with m/z from the inclusion list) was performed in the Orbitrap. The collision energy was set at 28% and resolution for the MS/MS was set at 35,000 for 200,000 ions with maximum filling times of 110 ms with an isolation width of 2.0 m/z. Data acquisition was done using Xcalibur version 4.3.73.11. and further analyzed on Skyline software\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e ( version 19.1.0.193).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry data analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIdentification of protein modified by Ub\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e. Raw files were analyzed with DIA-NN and the Uniprot human proteome database (version: March 21, 2020, \u0026minus;\u0026thinsp;75 776 entries). Briefly, the following parameters were entered in DIA-NN\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e: 2 miscleavages were allowed; fixed modification was carbamidomethylation on cysteine; enzyme was set as Trypsin/P. A maximum of 2 variable modifications were defined: carbamylation on lysines and N-term extremities. A mass tolerance of 20 ppm was used both for precursor and fragment ions. Identification value \"Precursor FDR\" was set to 0.01. Other settings include Peptide length range: 7\u0026ndash;30; Precursor charges range: 2\u0026ndash;4; precursor m/z range: 300\u0026ndash;1800; and finally, Fragment ion m/z range 200\u0026ndash;1800. The option \"Match between runs\" was also allowed. Following peptide identification, data were filtered using Prostar\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e: only proteins found in at least two out of the three replicates were considered for further analysis. Contaminants were removed and a VSN normalization within conditions was applied. Missing values on entire condition (MEC) were imputed with minimal value (first quantile) and partial observed value (POV) were imputed with SLSA. Detected intensities were transformed using the log\u003csub\u003e2\u003c/sub\u003e function and Z-scores were calculated for each protein in all three datasets. Figures featuring log\u003csub\u003e2\u003c/sub\u003e intensity and Z-scores were plotted using GraphPad software (GraphPad Software - version 9.0.0., the USA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMapping of Ub\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eand Ub diGly signature.\u003c/em\u003e Analysis of raw MS data was done using MaxQuant (version 1.6.17.0)\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Quantification was done with light (Lys\u003csub\u003e0\u003c/sub\u003eArg\u003csub\u003e0\u003c/sub\u003e) and medium (Lys\u003csub\u003e4\u003c/sub\u003eArg\u003csub\u003e6\u003c/sub\u003e) labels, and considering a trypsin digestion of the peptides with no cleavages on lysine or arginine before a proline. A maximum of two missed cleavages were allowed with methionine oxidation and protein N-terminal acetylation as variable modifications of proteins and carbamidomethylation as fixed modification. The maximum number of modifications allowed per peptide was set to 5. Mass tolerance was set to a maximum of 7 ppm for the precursor ions and 20 ppm for the fragment ions. Re-quantification of selected isotopic patterns was allowed to obtain ratios of all SILAC pairs. Second peptide search was also allowed. The minimum length of peptides to be considered for quantification was set to seven amino acids and the false discovery rate threshold set to 5%. The minimum number of peptides to be used for the identification of proteins was set to one but only proteins identified with two or more peptides were considered in further analysis. Protein quantification was calculated using both unique and razor peptides. A table containing all diGly signature sites detected by mass spectrometry was also generated. This file was used to determine the Log\u003csub\u003e2\u003c/sub\u003e(M/L) SILAC ratio for each identified site in order to identify sites specifically modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e. More, repartition of diGly sites per protein was analyzed for all detected sites, Ub\u003csup\u003eKEKS\u003c/sup\u003e specific sites and Ub specific sites. Finally, sequences of residues surrounding lysines specifically modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e (i.e., log\u003csub\u003e2\u003c/sub\u003e(M/L) \u0026lt; -1) were also retrieved from the table given by the MaxQuant software. These sequences measuring exactly 31 amino acids were later used in a Python script to study the environment around lysines modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e (please, refer to the paragraph \u0026ldquo;Analysis of the environment of Ub\u003csup\u003eKEKS\u003c/sup\u003e-modified lysines above).\u003c/p\u003e \u003cp\u003e \u003cem\u003eUb\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e:\u003cem\u003eUb equilibrium for PCNA\u0026rsquo;s modification.\u003c/em\u003e Identification and quantification of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e peptides was performed on Skyline software\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e (version 19.1.0.193). For quantification, the most intense fragment ions were used for all peptides: y8, y7, y6, y4, y3 and b3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e- Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The amount of Ub and Ub\u003csup\u003eKEKS\u003c/sup\u003e proteins were calculated using the light to heavy peptide ratio. Obtained ratios were plotted in GraphPad software (GraphPad Software - version 9.0.0., USA) for statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIdentification of proteins modified by Ub\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e \u003cem\u003evia the AviTag system.\u003c/em\u003e Detected intensities for each protein were transformed using the log\u003csub\u003e2\u003c/sub\u003e function to obtain datasets following Gaussian distributions. Z-score were then calculated for every data point. Proteins with a Z-score superior to the 90% percentile (0.9673 for CTL; 1.233 for Ub and 1.262 for Ub\u003csup\u003eKEKS\u003c/sup\u003e) within each data set was considered as significantly enriched. Two additional thresholds were also considered: Z-scores higher than the 95% percentile (1.389 for CTL; 1.857 for Ub and 1.824 for Ub\u003csup\u003eKEKS\u003c/sup\u003e) and Z-scores superior to the 99% percentile (2.085 for CTL; 3.018 for Ub and 2.965 for Ub\u003csup\u003eKEKS\u003c/sup\u003e) (n\u0026thinsp;=\u0026thinsp;4 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDistribution of SILAC ratios associated to diGly signature sites.\u003c/em\u003e Significance thresholds were arbitrarily set for depletion or enrichment of at least 2-folds. On Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, these values correspond to log\u003csub\u003e2\u003c/sub\u003e(M/L) = -1 and log\u003csub\u003e2\u003c/sub\u003e(M/L)\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1 (n\u0026thinsp;=\u0026thinsp;3 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003eComposition of the environment surrounding lysines modified by Ub\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e. Enrichment (or depletion) of specific amino acid was calculated by comparing frequency distributions of residues surrounding lysines modified by Ub\u003csup\u003eKEKS\u003c/sup\u003e and surrounding all lysines listed in the Uniprot database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Significance was determined by a Binomial test in Microsoft Excel with the following parameters: number of successes corresponds the occurrence of the residue in the Ub\u003csup\u003eKEKS\u003c/sup\u003e-modified lysines analysis; number of trials was set to 655; the probability was defined as proportion of the same residue at the same position found in the Uniprot\u0026rsquo;s lysines analysis; and finally cumulative was considered as true. A table containing of the calculated p-value for each amino acid and each position is available in Supp Table\u0026nbsp;5 (n\u0026thinsp;=\u0026thinsp;3 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003eNumber of modification sites per protein.\u003c/em\u003e For all modification sites identified across the human proteome and the ones specifically targeted by Ub\u003csup\u003eKEKS\u003c/sup\u003e, the number of modification sites per protein is displayed in Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The distribution between these 2 categories is similar and no further statistical analysis was performed (n\u0026thinsp;=\u0026thinsp;3 independent experiments)\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuantification of the Ub\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e:\u003cem\u003eUb ratio modifying PCNA.\u003c/em\u003e For each combination of plasmids, mean and standard deviations of measured Ub\u003csup\u003eKEKS\u003c/sup\u003e:Ub ratios are the following: PCNA-GFP\u0026thinsp;+\u0026thinsp;pcDNA3.1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:=\\:2.416\\times\\:{10}^{-2}\\:\\pm\\:0.011\\)\u003c/span\u003e\u003c/span\u003e; PCNA-GFP\u0026thinsp;+\u0026thinsp;Ub-HA \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:=\\:6.337\\:\\times\\:\\:{10}^{-3}\\:\\pm\\:0.005\\)\u003c/span\u003e\u003c/span\u003e; et PCNA-GFP\u0026thinsp;+\u0026thinsp;Ub\u003csup\u003eKEKS\u003c/sup\u003e-HA \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:=\\:11.34\\:\\pm\\:3.945\\)\u003c/span\u003e\u003c/span\u003e. No further statistical test was performed (n\u0026thinsp;=\u0026thinsp;3 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003eS phase progression monitoring.\u003c/em\u003e HeLa WT\u0026rsquo;s population showed \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:25.5\\pm\\:3.676\\:\\text{\\%}\\)\u003c/span\u003e\u003c/span\u003e of cells in late S phase at 4h and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:18.23\\pm\\:4.219\\:\\text{\\%}\\)\u003c/span\u003e\u003c/span\u003e of cells in G1 phase at 8h. On the other hand, population of HeLa 2.7 showed \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:20.73\\pm\\:3.262\\:\\text{\\%}\\)\u003c/span\u003e\u003c/span\u003e of cells in late S phase at 4h and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:9.82\\pm\\:4.473\\:\\text{\\%}\\)\u003c/span\u003e\u003c/span\u003e of cells in G1 phase at 8h. Finally, HeLa 4.3 populations showed \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:22.8\\pm\\:2.5\\:\\text{\\%}\\)\u003c/span\u003e\u003c/span\u003e of cells in late S phase at 4h and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:10.45\\pm\\:4.095\\:\\text{\\%}\\)\u003c/span\u003e\u003c/span\u003e of cells in G1 phase at 8h. Significance was determined by a Repeated Measures One-way ANOVA, followed by a Dunnett\u0026rsquo;s multiple comparisons post hoc test. At 4h, both comparison HeLa WT/HeLa 2.7 and HeLa WT/HeLa 4.3 revealed significant differences with p-values equal to 0.0025 and 0.0102 respectively. At 8h, these differences were exacerbated as p-values for both comparisons dropped below 0.0001 (n\u0026thinsp;=\u0026thinsp;3 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell cycle distribution analysis.\u003c/em\u003e Percentages of cells in G1, S and G2/M phases were measured and observed variations were assessed by a two-way ANOVA and Tukey\u0026rsquo;s multiple comparisons post hoc test. Comparison between HeLa WT and HeLa 2.7 cells resulted in nonsignificant p-values for all cell cycle phases (0.4744 for G0/G1 phase, 0.9561 for S phase and 0.6412 for G2/M phase). Comparison between HeLa WT and HeLa 4.3 cells led to the same conclusion (0.9782 for G0/G1 phase, 0.4307 for S phase and 0.8021 for G2/M phase)(n\u0026thinsp;=\u0026thinsp;3 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePCNA levels in total proteomes from wild-type and Ub\u003c/em\u003e \u003csup\u003e \u003cem\u003eKEKS\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eknockout cells.\u003c/em\u003e Differential abundance was assessed with a Limma t-test with slim pi0 calibration. Molecular levels were considered as significantly modulated if at least a 2-fold difference was observed and the corresponding p-value was lower than or equal to 0.01. Differential analysis of HeLa WT with HeLa 2.7 on one hand, and HeLa WT with HeLa 4.3 on the other hand, resulted in p-values of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:3.11\\times\\:{10}^{-2}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:2.61\\times\\:{10}^{-4}\\)\u003c/span\u003e\u003c/span\u003e respectively. However, since all observed differences were lower than to 2-fold difference, variation of PCNA\u0026rsquo;s cellular levels were ruled out as not significant (n\u0026thinsp;=\u0026thinsp;3 independent experiments, each read twice by the mass spectrometer).\u003c/p\u003e \u003cp\u003e \u003cem\u003eProcessivity of DNA polymerases.\u003c/em\u003e Replication speed in wild-type HeLa cells was equal to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:1.11\\pm\\:\\text{0,39}\\)\u003c/span\u003e\u003c/span\u003e kb/min. Absence of Ub\u003csup\u003eKEKS\u003c/sup\u003e lead to replication speeds of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:0.72\\pm\\:\\text{0,25}\\)\u003c/span\u003e\u003c/span\u003e kb/min and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:0.53\\pm\\:\\text{0,26}\\)\u003c/span\u003e\u003c/span\u003e kb/min for HeLa 2.7 and HeLa 4.3 cells respectively. Significance was determined using a One-way ANOVA, with a Dunnett\u0026rsquo;s multiple comparison post hoc test. Both comparison between HeLa WT and each Ub\u003csup\u003eKEKS\u003c/sup\u003e knockout clone resulted in a p-value smaller than 0.001 (n\u0026thinsp;\u0026gt;\u0026thinsp;150 measurements, distributed over 2 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSymmetry of replication forks.\u003c/em\u003e A symmetrical ratio was determined for each cell line: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:1.29\\:\\pm\\:0.4\\)\u003c/span\u003e\u003c/span\u003e for HeLa WT; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:1.37\\:\\pm\\:0.58\\)\u003c/span\u003e\u003c/span\u003e for HeLa 2.7 and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:1.55\\:\\pm\\:0.76\\)\u003c/span\u003e\u003c/span\u003e for HeLa 4.3. Significance was determined by a Kruskal-Wallis test, followed by Dunn\u0026rsquo;s multiple comparisons assay. Comparisons HeLa WT/HeLa 2.7 and HeLa WT/HeLa 4.3 led to p-values equal to 0.2884 and 0.0156 respectively (n\u0026thinsp;\u0026gt;\u0026thinsp;75 measures per samples, distributed over 2 independent experiments).\u003c/p\u003e \u003cp\u003e \u003cem\u003eDistribution of replication structures at active replication forks.\u003c/em\u003e Impacts of the presence or absence of Ub\u003csup\u003eKEKS\u003c/sup\u003e on the different replication structures were assessed using a Chi-square test. Since the analysis was performed on large populations, an additional Cram\u0026eacute;r\u0026rsquo;s V test was also done to evaluate potential bias. Parameters and results of both statistical tests are available in Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec (n\u0026thinsp;=\u0026thinsp;782 for HeLa WT; 665 for HeLa 2.7 and 804 for HeLa 4.3; all distributed over 2 independent experiments).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eJ.F. is the recipient of a \u0026ldquo;Fonds de Recherche du Qu\u0026eacute;bec \u0026ndash; Sant\u0026eacute;\u0026rdquo; (FRQS) studentship (grant number #313345). Funding was provided from the Canadian Institutes for Health Research, grant number #398925 to F.-M.B. F.-M.B. is a FRQS Senior scholar (award number 281824). X.R. is a recipient of a Canada Research Chair in Functional Proteomics and Discovery of Novel Proteins. X.R. and F.M.B. are members of the FRQS-funded \u0026ldquo;Centre de Recherche du CHUS\u0026rdquo;.\u003c/p\u003e\n\u003ch2\u003eAuthor contribution\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eConceptualization: J.F., X.R. and F-M.B.; Methodology: J.F., A.M. and F-M.B.; Formal analysis: J.F. and J.R.; Investigation: J.F., J.R., G.M., D.L. and M-L.D.; Programming: J.F.; Writing \u0026ndash; original draft: J.F.; Writing \u0026ndash; review and editing: J.F., X.R. and F-M.B.; Supervision: X.R. and F-M.B.; Project administration: F-M.B.; Funding acquisition: F-M.B. All authors have read and agreed to the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eMaterials\u0026nbsp;and Correspondence\u003c/h2\u003e\n\u003cp\u003eCorrespondence and material requests should be addressed to Fran\u0026ccedil;ois-Michel Boisvert at [email protected]\u003c/p\u003e\n\u003ch2\u003eImages license\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eSome figures used in this article were generated using the SMART medical art platform from Servier (https://smart.servier.com/). Raw images are licensed under a Creative Commons Attribution 3.0 Unported License. More information on this license is available at https://creativecommons.org/licenses/by/3.0/\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData\u0026nbsp;availability\u003c/h2\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been uploaded on the ProteomeXchange Consortium via the PRIDE\u003csup\u003e65\u003c/sup\u003e partner repository under the dataset identifier PXD061769.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCode availability\u003c/h2\u003e\n\u003cp\u003ePython scripts used in this article are available in the GitHub repository at https://github.com/fmboisvert/KAnalysis, under the Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) license.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez-Maga\u0026ntilde;a A, Blanco FJ (2020) Human PCNA structure, function and interactions. 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Mass Spectrom Rev 39:229\u0026ndash;244\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"UbKEKS, Proliferating Cell Nuclear Antigen (PCNA), Post-Translational Modification (PTM), DNA fiber combing assay, Data-Independent Acquisition (DIA), Data-Dependent Acquisition (DDA), SILAC labelling, PRM quantification, AviTag","lastPublishedDoi":"10.21203/rs.3.rs-6515396/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6515396/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProliferating Cell Nuclear Antigen (PCNA) is a pivotal regulator of DNA replication and repair, orchestrating protein recruitment through its binding domains and post-translational modifications (PTMs). Lysine 164 (K164) of PCNA serves as a critical molecular switch modulated by ubiquitin (Ub) and ubiquitin-like proteins. Here, we identify the ubiquitin variant Ub\u003csup\u003eKEKS\u003c/sup\u003e as a new PCNA regulator, demonstrating its covalent modification of K164 and quantifying the Ub\u003csup\u003eKEKS\u003c/sup\u003e-to-Ub modification ratio. Loss of Ub\u003csup\u003eKEKS\u003c/sup\u003e results in delayed S-phase progression without altering PCNA levels or nuclear localization. However, DNA fiber combing assays reveal a significant reduction in DNA polymerase processivity in Ub\u003csup\u003eKEKS\u003c/sup\u003e-deficient cells, indicating its role in replication efficiency. At a whole-cell scale, the mapping by mass spectrometry of diGly remnants after trypsin digestion, demonstrate that modifications by Ub\u003csup\u003eKEKS\u003c/sup\u003e or Ub do not compensate one another, due to key differences regarding amino acids surrounding modified lysines. Ultimately, our findings establish Ub\u003csup\u003eKEKS\u003c/sup\u003e as a distinct key modulator of PCNA function, expanding the repertoire of PTMs that influence DNA replication dynamics. These insights pave the way for further exploration of Ub\u003csup\u003eKEKS\u003c/sup\u003e as a regulator of genome stability and cell cycle regulation.\u003c/p\u003e","manuscriptTitle":"The ubiquitin variant UbKEKS modifies PCNA to enhance DNA polymerase processivity during replication","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 11:29:33","doi":"10.21203/rs.3.rs-6515396/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e8cee91-7794-4c83-8de4-8f414a90a735","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48252034,"name":"Biological sciences/Cell biology/Post-translational modifications/Ubiquitylation"},{"id":48252035,"name":"Biological sciences/Cell biology/Cell division/DNA replication/DNA synthesis"},{"id":48252036,"name":"Biological sciences/Biochemistry/Proteomics/Protein\u0026#x2013;protein interaction networks"},{"id":48252037,"name":"Biological sciences/Biological techniques/Proteomic analysis"},{"id":48252038,"name":"Biological sciences/Cell biology/Cell division/Checkpoints"}],"tags":[],"updatedAt":"2025-06-05T11:55:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 11:29:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6515396","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6515396","identity":"rs-6515396","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}