The Mms22-Rtt107 axis dampens the DNA damage checkpoint by reducing the stability of the Rad9 checkpoint mediator

The Mms22-Rtt107 axis dampens the DNA damage checkpoint by reducing the stability of the Rad9 checkpoint mediator | 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 Mms22-Rtt107 axis dampens the DNA damage checkpoint by reducing the stability of the Rad9 checkpoint mediator Xiaolan Zhao, Bingbing Wan, Danying Guan, Shibai Li, Tzippora Chwat-Edelstein This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4417144/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The DNA damage checkpoint is a highly conserved signaling pathway induced by genotoxin exposure or endogenous genome stress. It alters many cellular processes such as arresting the cell cycle progression and increasing DNA repair capacities. However, cells can downregulate the checkpoint after prolonged stress exposure to allow continued growth and alternative repair. Strategies that can dampen the DNA damage checkpoint are not well understood. Here, we report that budding yeast employs a pathway composed of the scaffold protein Rtt107, its binding partner Mms22, and an Mms22-associated ubiquitin ligase complex to downregulate the DNA damage checkpoint. Mechanistically, this pathway promotes the proteasomal degradation of a key checkpoint factor, Rad9. Furthermore, Rtt107 binding to Mms22 helps to enrich the ubiquitin ligase complex on chromatin and target the chromatin-bound form of Rad9. Finally, we provide evidence that the Rtt107-Mms22 axis operates in parallel with the Rtt107-Slx4 axis, which displaces Rad9 from chromatin. We thus propose that Rtt107 enables a bifurcated “anti-Rad9” strategy to optimally downregulate the DNA damage checkpoint. Biological sciences/Molecular biology/DNA damage and repair/DNA damage checkpoints Biological sciences/Molecular biology/DNA damage and repair/DNA damage response Biological sciences/Genetics/Genomic instability DNA damage checkpoint checkpoint dampening Rtt107 Mms22 Slx4 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The highly conserved DNA damage checkpoint (DDC) is a critical component of the genome stress response 1 – 3 . When cells suffer from increased burdens of genome lesions caused by genotoxins or endogenous sources, the DNA damage sensor proteins can recruit apical DDC kinases to DNA lesion sites leading to their subsequent activation 4 , 5 . DDC mediator proteins can then amplify the genome stress signals by recruiting downstream transducer kinases, which can be phosphorylated and activated by the apical DDC kinases 6 – 8 . The activated transducer kinases can diffuse to various cellular compartments and phosphorylate hundreds of substrates 9 . Such a large-scale phosphoproteomic transformation can induce a myriad of cellular changes. These include cell cycle arrest to provide more time for DNA repair, increased ability to repair, and transcriptional alterations, among many other consequences 3 , 10 . While DDC-induced changes are beneficial for cells to cope with stress temporarily, long-term survival also depends on the ability to dampen the checkpoint once stress is dealt with or when stress becomes persistent 11 – 13 . Checkpoint dampening can permit cell cycle resumption and thus the chance of continued growth, as well as access to DNA repair mechanisms operating in different cell cycle phases, among other benefits 11 – 13 . However, the various strategies employed to dampen DDC remain to be discovered. DDC dampening has thus far been mostly examined in the model organism budding yeast. Initial studies have focused on protein phosphatases that can reverse some of the phosphorylation changes elicited by the DDC kinases 11 . However, phosphatases alone are insufficient for DDC dampening, and cells also need to reduce the continuous emission of DDC signals generated through the chains of the DNA damage sensors, apical DDC kinases, checkpoint mediators, and transducer kinases. Examples of phosphatase-independent strategies to downregulate DDC include displacing checkpoint factors from chromatin 14 – 18 . Whether and how additional pathways can reduce DDC signaling remain to be elucidated. Here we report a strategy of DDC dampening mediated by proteasomal degradation of a key checkpoint mediator protein Rad9 in yeast. Rad9 associates with DNA lesion sites in response to genotoxin treatment through binding to nucleosomes containing γH2A that demarcate damage domains and methylated H3K79 19, 20 . Rad9 can then recruit the transducer kinase Rad53 in preparation for Rad53 phosphorylation and subsequent activation by the main apical DDC kinase, Mec1 21, 22 . As Rad53 is responsible for a large majority of DDC-induced phosphorylation events in yeast, regulating its upstream enabler, Rad9, can be a key step to downregulate DDC. Indeed, a scaffolding complex composed of the Rtt107 and Slx4 proteins was shown to compete with Rad9 for binding to nucleosomes, thus reducing Rad9’s association with DNA lesion sites 14 , 23 . Interestingly, Rtt107 can additionally bind to another scaffold protein Mms22 24–26 . Mms22 was shown to serve as a substrate adaptor of the cullin ubiquitin E3 complex, which also contains the E3 subunit Rtt101 and the Mms1 subunit (Rtt101 Mms 1 E3) 27 . While the Mms22-Rtt101 Mms 1 E3 complex is known to protect genome stability and support cell growth when facing genotoxins, its functional mechanisms are not fully understood 28 . We show here that Rtt107 binding to Mms22 as well as the Rtt101 Mms 1 E3 are required for Rad9 degradation. We also demonstrate that Mms22 and Slx4 act in parallel to achieve a more effective “anti-Rad9” outcome during DDC recovery. As such, Rtt107 enables a two-pronged strategy to downregulate Rad9-mediated DDC via collaborating with its two binding partners. RESULTS Rtt107 association with Mms22 prevents genotoxin sensitivity and prolonged checkpoint Both Rtt107 and Mms22 are important for genome stability; however, the biological functions of their interaction have been unclear. To address this question, we examined a separation-of-function allele of Mms22 that specifically disrupts the Rtt107 binding without affecting other known interactions 29 . Given that Rtt107 and Mms22 each have multiple binding partners, this allele provides a valuable reagent to define the biological functions of their interaction 25 , 28 , 30 . High-resolution structure has revealed that Rtt107 binds to the N-terminal Rtt107-interaction-motif (RIM) of Mms22 29 . Further, alanine substitution of two residues (D13 and Y33) within Mms22’s RIM ( mms22 RIM ) disrupts the Mms22 and Rtt107 interaction (Fig. 1a) 29 . mms22 RIM supports the wild-type level of protein expression and interactions with other binding partners, such as the Mms1 protein that associates with the C-terminal region of Mms22 (Supplementary Fig. 1a) 25 , 29 . We confirmed here that mms22 RIM led to sensitivity to the DNA methylation agent MMS (methyl methanesulfonate) and further showed that it also caused sensitivity to the Top1 trapping compound CPT (camptothecin) and the replication fork blocking agent HU (hydroxyurea) (Fig. 1b). Compared with mms22 RIM , mms22∆ led to greater sensitivity to MMS, CPT, and HU, as well as slower growth (Fig. 1b). This agrees with the notion that mms22 RIM is a separation-of-function allele affecting the Mms22-Rtt107 binding without interfering with other roles of Mms22. Sensitivity to several genotoxins raised the possibility that mms22 RIM may affect common processes during different genotoxin responses such as the DNA damage checkpoint. To test this notion and determine the underlying causes of the genotoxin sensitivity exhibited by mms22 RIM cells, we focused on MMS treatment since DDC has been well examined in this condition. Specifically, we examined how synchronized cells moved through the cell cycle in a regimen that allows monitoring of both the initial response to MMS and the recovery. Briefly, cells synchronized in G1 were released to the cell cycle in the presence of MMS for 45 minutes, and then allowed to recover in MMS-free media (Fig. 1c, right). As expected, wild-type cells progressed slowly through S phase and were able to enter the second cell cycle after MMS washout (Fig. 1c). We also examined Rad53 activation, a marker for DDC signaling. The F9 antibody is widely used to detect Rad53 activation as it specifically recognizes the phosphorylated but not unmodified form of Rad53, although the antibody also binds to a nonspecific band only in G1 cells 31 . As previously reported, we observed that in wild-type cells, Rad53 activation was induced in S phase when cells were treated with MMS (30–45 min) and gradually diminished during the recovery phase (Fig. 1d and Supplementary Fig. 1b). In contrast to wild-type cells, mms22 RIM cells delayed the re-entry into the second G1 phase after MMS washout, though showed no obvious defects during S phase progression in the presence of MMS (Fig. 1c). Concomitantly, Rad53 phosphorylation induced by MMS failed to decline during the recovery phase (Fig. 1d and Supplementary Fig. 1b). The results from these two assays are consistent with each other and suggest that mms22 RIM cells exhibit persistent DDC during the recovery phase, which can delay entry to the next cell cycle. Genotoxin sensitivity associated with the loss of the Mms22-Rtt107 interaction is rescued by reducing the DNA damage checkpoint Persistent checkpoint can be caused by delayed genome replication and repair or by reduced ability to dampen the checkpoint itself. The two scenarios differ in how cells would respond to reduced levels of DDC. In the first scenario, cell viability would suffer when checkpoint function is weakened, because cells rely on optimal checkpoint to complete DNA replication and repair. In contrast, cells in the second scenario could show better survival upon checkpoint weakening. Based on this rationale, we tested how mms22 RIM cells responded to reduced DDC levels conferred by mild alleles of the checkpoint mediator protein Rad9 or Ddc1. The two mutants used, namely rad9-K1088M and ddc1-T602A , contain single point mutations that reduce Rad9 binding to γH2A and Ddc1 binding to another checkpoint factor Dpb11 20, 32 . These studies have shown that rad9-K1088M and ddc1-T602A mildly reduce DDC. Significantly, we found that either allele conferred strong rescue of the MMS sensitivity of mms22 RIM cells (Fig. 2a). While the DNA damage checkpoint can operate throughout the cell cycle, cells also employ the DNA replication checkpoint (DRC) during S phase 3 . We next examined whether mild hypomorphic DRC mutants could affect the MMS sensitivity of mms22 RIM cells. We tested two well-characterized alleles affecting either the Mrc1 mediator protein of the DRC pathway, namely mrc1-AQ , or mec1-100 that is specifically defective in DRC 33 , 34 . As shown in Fig. 2b, neither allele affected the MMS sensitivity of mms22 RIM cells. Together, these data suggest that defects of mms22 RIM cells can be rescued by reducing DDC, but not DRC, functions. Collectively, the genetic findings raise the possibility that Mms22 binding to Rtt107 contributes to the downregulation of DDC but not DRC, and that this role can be partly responsible for the genotoxin sensitivity caused by the loss of the Mms22-Rtt107 interaction. Persistent checkpoint in mms22-RIM cells is rescued by rad9-K1088M and ddc1-T602A To further test the above hypothesis, we examined whether the suppression of MMS sensitivity of mms22 RIM cells by the DDC mutants is associated with a correction of the persistent checkpoint seen in mms22 RIM cells. We used a similar experimental scheme as depicted in Fig. 1c to monitor cell cycle progression and Rad53 activation, except a longer period of recovery was examined. Similar to observations described above (Fig. 1c), while wild-type cells were able to enter the next cell cycle after recovery from MMS treatment, mms22 RIM delayed the entry (Fig. 2c and Supplementary Fig. 2a). This delay was quantified for two late time points (260 and 300 min; Fig. 2d). Significantly, this delay was improved by either rad9-K1088M or ddc1-T602A (Fig. 2c and Supplementary Fig. 2a). Compared with mms22 RIM , more cells in the mms22 RIM rad9-K1088M or mms22 RIM ddc1-T602A double mutants exited G2/M and entered the next G1 phase between 200 to 300 minutes, with the most prominent differences seen at 260 and 300 minutes (Fig. 2d). rad9-K1088M and ddc1-T602A behaved similarly to wild-type cells, reflecting redundancy among DDC mediator functions (Figs. 2c and 2d, Supplementary Fig. 2a). Importantly, rad9-K1088M or ddc1-T602A also reduced the persistent Rad53 activation seen in mms22 RIM cells (Fig. 2e and Supplementary Fig. 2b). Collectively, the suppression of prolonged Rad53 activation and cell cycle arrest seen in mms22 RIM by rad9-K1088M or ddc1-T602A provides further evidence that Mms22 binding to Rtt107 plays a role in dampening the DNA damage checkpoint. Mms22 and Slx4 act in parallel to dampen the DNA damage checkpoint Rtt107 has an established role in DDC dampening through pairing with Slx4 14, 23 . Rtt107 employs the same surface to engage with short motifs within Slx4 or Mms22, engendering mutually exclusive pairwise interactions 29 . A separation-of-function allele, slx4 TTS (T423, T424, and S567A), has been constructed that specifically abolishes the Slx4-Rtt107 interaction without affecting protein level or binding to other known partners, such as the Slx1 nuclease 29 . We thus asked how the Mms22-Rtt107 mediated effect on DDC recovery is functionally related to that mediated by Slx4-Rtt107. Applying the experimental scheme described in Fig. 2A, we found that slx4 TTS led to a more pronounced delay in late S phase compared with mms22 RIM (80 and 120 min; Fig. 3a). In contrast, slx4 TTS showed a milder delay in re-entry into the next cell cycle compared with mms22 RIM (240 and 300 min; Fig. 3a). These observations raised the possibility that the two Rtt107 interactors may differentially affect DDC at different stages of the cell cycle. Significantly, the mms22 RIM slx4 TTS double mutant exhibited more severe defects in exiting the G2/M arrest in the first cell cycle than either single mutant (Fig. 3a). At the end of the time course (300 min), the double mutant showed significantly fewer cells exited the G2/M phase than either single mutant (Fig. 3a). A similar additive effect was seen when assaying for the active form of Rad53. The mms22 RIM slx4 TTS double mutant showed higher levels of active Rad53 than either single mutant at the two last time points of the time course (240 and 300 min; Fig. 3b). Results from cell cycle analysis and active Rad53 forms can be best explained by that Mms22-Rtt107 and Slx4-Rtt107 complexes acting in different pathways to downregulate the DNA damage checkpoint. Additional evidence supports the independence of Mms22- and Slx4-mediated functions Further supporting the functional independence of Mms22 and Slx4, we found that the double mutant of mms22 RIM slx4 TTS led to stronger MMS sensitivity than either single mutant (Fig. 3c). The additive effect of mms22 RIM and slx4 TTS was also seen when assaying the stability of the repetitive ribosomal DNA (rDNA) locus and of a non-rDNA locus during growth, suggesting that their independence is a general feature (Supplementary Fig. 3). We showed that each single mutant caused a 4 to 5-fold increase in marker loss at rDNA, while the mms22 RIM slx4 TTS double mutant led to a 10-fold increase (Supplementary Fig. 3a). Similarly, in the gross chromosome rearrangement (GCR) assay that assesses the stability of a non-rDNA locus, we detected a 59-fold increase of marker loss in the mms22 RIM slx4 TTS double mutant and a 16 to 20-fold increase in the corresponding single mutants (Supplementary Fig. 3b). These genetic data are in line with the results from two cell cycle checkpoint assays described above as well as previous biochemical findings that Mms22 and Slx4 partner with Rtt107 in a mutually exclusive manner 29 . Together, they strongly suggest that Mms22 and Slx4 represent parallel pathways in regulating both the DNA damage checkpoint and genomic stability. While our data support the functional independence of Mms22 and Slx4, the two proteins have the potential to indirectly affect each other due to sharing a common partner, Rtt107, that facilitates the chromatin recruitment of both 29 . To examine this possibility, we asked if Mms22 and Slx4 affect each other’s chromatin association. We queried how the loss of the Slx4-Rtt107 interaction could affect Mms22 chromatin association and vice versa. We confirmed the reported findings that mms22 RIM and slx4 TTS each reduced its own chromatin association (Fig. 3d). In contrast, mms22 RIM led to an increase in Slx4 level on chromatin while slx4 TTS showed no effect on the chromatin association of Mms22 (Fig. 3d and Supplementary Fig. 3c). These results suggest that the checkpoint dampening defects seen for mms22 RIM cells are not due to an indirect effect of reducing Slx4 chromatin association, and vice versa. This notion is consistent with data describing the additive effect of mms22 RIM and slx4 TTS shown above (Figs. 3a-3c); together they suggest that Mms22 and Slx4 can work in parallel pathways with each collaborating with Rtt107 (Fig. 3e). The Mms22-Rtt107 interaction and the Rtt101 E3 promote Rad9 degradation We next investigated the mechanisms by which the Mms22-Rtt107 interaction can promote DDC dampening. As Mms22 is a subunit of the Rtt101 Mms 1 ubiquitin E3 27 , a likely means for it to downregulate the checkpoint is through protein degradation. Given the strong genetic suppression of mms22 RIM by rad9 and ddc1 mutant alleles (Figs. 2a and 2c-2e, Supplementary Fig. 2), we examined the stability of these two proteins. We performed a standard procedure that utilizes cycloheximide (CHX) to block new protein synthesis, thus allowing the monitoring of protein stability during a time course. We first examined Ddc1 and Rad9 during normal growth. While the Ddc1 protein level showed minimal changes during an eight-hour time course, the Rad9 protein level exhibited a strong reduction over time (Figs. 4a and 4b, Supplementary Fig. 4a). Importantly, Rad9 instability is improved by the removal of Rtt101 or Rtt107, and by mms22 RIM (Fig. 4a). Similar improvement was also seen in mms22∆ and mms1∆ cells (Supplementary Fig. 4b). Rad9 protein level quantification based on at least two biological isolates per genotype showed that all examined mutants stabilized Rad9 levels to a similar degree (Fig. 4b and Supplementary Fig. 4c). In contrast to this group of mutants, Rad9 degradation was not affected by the lack of Slx4 (Supplementary Fig. 4d). We thus conclude that the Rtt107-Mms22-Rtt101 Mms 1 axis but not the Rtt107-Slx4 axis affects Rad9 protein stability. As mutants of Rtt107, Mms22, and Rtt101 did not fully stabilize Rad9, additional means also exist to promote Rad9 degradation. We next monitored Rad9 stability when cells were treated by MMS. Again, we observed Rad9 degradation in wild-type cells and this was reduced upon the removal of Rtt107, Rtt101, and in mms22 RIM cells (Figs. 4c and 4d). This group of mutants also showed persistent Rad9 phosphorylation, which is catalyzed by the Mec1 kinase and serves as a marker for DDC activation (Fig. 4c) 21 , 35 . These results are consistent with data presented above and further support the conclusion that the Mms22-Rtt107 interaction is required for degrading Rad9. Collectively, our findings suggest that the Mms22-Rtt107 interaction as well as the Rtt101 Mms 1 E3 are partly responsible for Rad9 degradation. Rad9 degradation is mediated by proteasomes To further test the above notion, we asked whether Rad9 protein instability is mediated by the proteasomes. If Mms22 and Rtt107 collaborate with the Rtt101 Mms 1 E3 in Rad9 degradation, we would expect that blocking proteasomal functions can hinder Rad9 degradation. We treated cells with MG132 that inhibits proteasomal activity, and with CHX that blocks new protein synthesis. In the control DMSO treatment, Rad9 protein level reduced during a four-hour time course (Fig. 5a). However, the Rad9 protein was stabilized in the presence of MG132 (Fig. 5a). This result suggests that Rad9 degradation is mediated by proteasomes. This conclusion is in line with the involvement of the Rtt101 Mms 1 E3 in Rad9 degradation in cells. rad9-K1088M rescues the MMS sensitivity caused by the loss of Rtt101 and Mms1 We next examined the functional significance of the Rtt101 Mms 1 E3’s involvement in Rad9 degradation. If this role is important for cell survival in genotoxins, we would expect that as seen for mms22 RIM mutant, genotoxin sensitivity caused by the loss of the Rtt101 Mms 1 E3 could be rescued by a mildly defective Rad9 allele. Indeed, we found that rad9-K1088M greatly increased the viability of either rtt101∆ or mms1∆ mutant cells on media containing MMS, CPT, or HU (Fig. 5b). This data provides evidence that like Mms22, Rtt101 Mms 1 E3’s involvement in Rad9 degradation can also be important for cellular survival in the face of genotoxins. Mms22-Rtt107 interaction helps the Rtt101 Mms 1 E3 associate with chromatin and regulating Rad9 stability on chromatin Our data thus far suggest that Rtt107 dampens DNA damage checkpoint signaling through binding to Mms22, in addition to binding to Slx4. Previous studies have shown that Rtt107 helps to recruit both Mms22 and Slx4 to chromatin via its ability to recognize γH2A 14 , 29 , 36 , 37 . These findings raise the possibility that Rtt107 binding to Mms22 may help the chromatin association of the Rtt101 Mms 1 E3 for degrading Rad9 on chromatin. We tested the above notion first by examining the chromatin association of Rtt101 and Mms1 when the Mms22-Rtt107 interaction is disrupted by mms22 RIM . We used a well-established chromatin fraction method to separate chromatin and soluble fractions 38 . We found that mms22 RIM not only reduced its own chromatin association but also lessened those for Rtt101 and Mms1 (Fig. 6a). We moved on to monitor Rad9 levels on chromatin and found that mms22 RIM cells exhibited an increased amount of Rad9 on chromatin (Fig. 6b), suggesting that the Mms22-Rtt107 interaction is required for Rad9 loss from chromatin. Importantly, in the presence of CHX that allows the examination of protein degradation, we found that degradation of Rad9 in the chromatin fraction was largely blocked in mms22 RIM cells (Fig. 6c). The Rad9 stabilization effect conferred by mms22 RIM is much stronger for the chromatin pools of Rad9 compared with the whole cell extract (WCE) pool of Rad9 (Fig. 6c), suggesting that the main effect of Mms22-Rtt107 stems from the regulation of the stability of Rad9 in the chromatin fraction. Collectively, these data provide evidence that the Mms22-Rtt107 interaction is important for the chromatin association of the Rtt101 Mms 1 E3 and degrading Rad9 in the chromatin fraction. Mms22-Rtt107 binding does not affect the Rad53-Asf1 or Rtt107-Dpb11 association The Rtt101 Mms 1 E3 was previously shown to collaborate with Asf1 in down-regulating DDC when cells suffer from two double-strand DNA breaks (DSBs) 39 . The proposed model suggests that Rtt101 Mms 1 E3 may help Asf1 binding to Rad53, an interaction that could disfavor Rad53 phosphorylation thus leading to reduced DDC levels 39 . To discern if the Mms22 collaboration with Rtt101 Mms 1 during DDC dampening might be related to Asf1-Rad53 association, we examined this interaction by co-immunoprecipitation. We found that the amount of Asf1 recovered from Rad53 co-immunoprecipitation was similar between mms22 RIM and wild-type cells, in both normal growth and MMS treated conditions (Supplementary Fig. 5a). This result suggests that the effect of the Mms22-Rtt107 interaction on DDC dampening is not via regulating the Asf1-Rad53 interaction. Finally, considering that Rtt107 also associates with another checkpoint factor, Dpb11 23 , we asked whether this interaction is perturbed in mms22 RIM cells. We found that while mms22-RIM disrupted the Rtt107-Mms22 interaction, it did not affect the Rtt107-Dpb11 association in the presence or absence of MMS treatment (Supplementary Fig. 5b). These results suggest the DDC recovery role of the Rtt107-Mms22 interaction is not mediated by altering the Rtt107-Dpb11 interaction. DISCUSSION Cellular survival of genotoxin treatments depends on the induction of the DNA damage checkpoint as well as its subsequent downregulation. While many studies have examined DDC induction, DDC downregulation is less understood. Here we describe a DDC dampening strategy that is mediated by the degradation of a central DDC mediator protein, Rad9, in yeast. We identified the players that contribute to this regulation, including the scaffold protein Rtt107, the Mms22 substrate adapter of the Rtt101 Mms 1 ubiquitin E3, and the ubiquitin E3 itself. Results from genetic suppression and checkpoint assays strongly support the conclusion that the Mms22-Rtt107 interaction provides an important means to downregulate DDC. Further, biochemical evidence suggests that Mms22 and Rtt107 collaborate with the Rtt101 Mms 1 E3 to facilitate proteasome-mediated degradation of Rad9. Interestingly, this regulation was more prominently seen for the chromatin pool of Rad9 compared with the non-chromatin pool. The preferential effect can be explained by our finding that the Mms22-Rtt107 interaction promotes the chromatin association of the Rtt101 Mms 1 E3. Based on these data, we propose a model wherein Rtt107 recruits the Mms22-containing Rtt101 Mms 1 E3 to chromatin to facilitate Rad9 degradation (Fig. 6d). Chromatin targeting of the Mms22-containing E3 could be achieved through previously identified bifurcated interactions of Rtt107, with its tetra-BRCT domain binding to Mms22 and additional binding sites recognizing γH2A and the phosphorylated H4T80 mark 29 , 36 , 40 . Finally, we provide evidence to support the notion that the Rtt107-Mms22-Rtt101 Mms 1 E3 axis acts in parallel with the Rtt107-Slx4 axis that competes with Rad9 for binding to nucleosomes. As such, Rtt107 could coordinate a two-pronged strategy that exploits distinct anti-Rad9 effects to downregulate DDC (Fig. 6d). Rtt107 is required for chromatin targeting of both Mms22 and Slx4, which compete for the same binding site on the Rtt107 tetra-BRCT domain 29 . Despite the competition, both the Mms22-Rtt107 and Slx4-Rtt107 complexes are detected in cells 26 . Thus, Rtt107 is likely present in sufficient amounts to allow the formation of the two complexes. In line with this notion, the loss of Slx4-Rtt107 binding had no effect on the abundance of Mms22 on chromatin (Fig. 3d and Supplementary Fig. 3c). However, disrupting the Mms22-Rtt107 association increased Slx4 levels on chromatin (Fig. 3d and Supplementary Fig. 3c). These observations suggest a unidirectional modulation of the dynamics of the Rtt107 interactome that is worthwhile for further testing in the future. Most relevant here, despite showing increased chromatin association of Slx4, which could favor DDC downregulation, mms22 RIM cells nevertheless showed persistent checkpoint (Fig. 3d and Supplementary Fig. 3c). These data support our conclusion that the DDC dampening defect caused by mms22 mutants is not due to lessening Slx4 chromatin association and vice versa. Rather, Mms22 and Slx4 each employ a different strategy to downregulate the checkpoint. While the Slx4-Rtt107 axis can disfavor Rad9 binding to chromatin 14 , 23 , our result suggests that the Mms22-Rtt107 axis regulates Rad9 degradation. Interestingly, while mms22 RIM led to a general increase in Rad9 stability, it had a more prominent effect on the chromatin pool of Rad9, which is required for DDC signaling (Fig. 6c). This makes it possible that targeting this pool of Rad9 can make the Mms22-Rtt107 axis more efficient in downregulating the checkpoint. We note that we cannot exclude additional roles of Mms22-Rtt107 in affecting Rad9 association with chromatin. Nonetheless, since Slx4 did not affect Rad9 stability, regulation of Rad9 stability appears to be rather specific to the Mms22-Rtt107 axis and does not reflect a general feature of DDC dampening factors. Using a Slx4 mutant specifically abolishing its binding to Rtt107, our data strengthened the previous conclusions regarding the role of Slx4 and Rtt107 in checkpoint dampening. Further, we show that the Slx4-Rtt107 interaction had a stronger effect when the Mms22-Rtt107 interaction was lost (Figs. 3a and 3b). In addition, slx4 TTS cells showed slower S phase progression compared with wild-type cells. This effect can be related to findings that Slx4 and Rtt107 associate with chromatin behind the replication fork to help replication in MMS conditions 37 . In contrast, mms22 RIM had no obvious defect in S phase progress but delayed exiting from the G2/M phase and increased levels of Rad53 activation (Figs. 3a and 3b). These observations provide additional support for the conclusion that Rtt107 can participate in two DDC regulatory pathways involving either Mms22 that mainly affects G2/M exit or involving Slx4 that has roles in controlling S phase progression. The proteins examined here, including Rtt107, Mms22, Rtt101 Mms 1 and Slx4 are all multi-functional and each has several binding partners 25 , 28 , 30 , 41 . In addition to our findings here, the Rtt101 Mms 1 E3 has been suggested to downregulate DDC via regulating the Asf1-Rad53 interaction 39 . Using the mms22 RIM separation-of-function allele, we showed that the Rtt107-Mms22 collaboration with Rtt101 Mms 1 E3 does not affect the Asf1-Rad53 association (Supplementary Fig. 5a). These data provide evidence that the Rtt101 Mms 1 E3 has at least two roles in checkpoint dampening, one through Rad9 degradation and the other via regulating the Asf1-Rad53 interaction. We also found that mms22 RIM did not affect the Rtt107 interaction with another checkpoint mediator Dbp11 known to also bind to Slx4 (Supplementary Fig. 5b). These data highlight the importance of using separation-of-function alleles in clarifying the role(s) of complex protein-protein interactions employed in DDC and genome maintenance. Our data suggest that additional ubiquitin E3(s) can also help proteasome-mediated degradation of Rad9. It is common that one substrate is subjected to multiple E3 regulations to ensure their optimal degradation. Future studies aiming to identify additional factors involved in Rad9 degradation can expand our understanding of protein-level control of this key checkpoint mediator. Though our work focuses on the role of the Mms22-Rtt107 interaction in DNA damage conditions, we found that disruption of this interaction by mms22 RIM also affects genome maintenance during growth. In particular, mms22 RIM led to increased genomic instability at both rDNA and non-rDNA sites in the absence of genotoxin and showed additive effect with slx4 TTS (Supplementary Figs. 3a and 3b). This genetic finding corroborates the biochemical data that Rad9 stability is similarly regulated by Mms22 during growth and under genotoxin treatment, suggesting that Rad9 is a general client for the Mms22-Rtt101 Mms 1 E3 and its degradation is not triggered by DDC (Figs. 4a and 4b). Together, these data raise the possibility that the genome-protecting roles of the Rtt107-Mms22 axis during growth are also related to limiting Rad9 levels. It is currently unclear how limiting Rad9 levels during growth is beneficial to cells. Given that the Mec1 kinase shows local activation but does not trigger global DDC during normal S phase, we speculate that Rad9 degradation may help restrain Mec1 activation to a local level, thus preventing unnecessary and harmful full-on DDC 3 , 42 . In this scenario, DDC dampening mechanisms may not only be critical for long-term survival in genomic stress conditions but could also help manage genome stability during growth. Future studies to explore these possibilities can provide a fuller picture to better unite DDC regulation in both conditions. Given the conservation of the protein factors examined here, results of our study can stimulate the studies of how higher eukaryotes can utilize protein degradation as a tool to downregulate checkpoint, and of the functional relevance of such regulation in tumorigenesis and during normal development. METHODS Yeast strains and genetic manipulation. Yeast strains used in this work are listed in Table S1 and they are derivatives of W1588-4C, a RAD5 variety of W303 ( MATa ade2-1 can1-100 ura3-1 his3-11,15 leu2-3,112 trp1-1 rad5-535 ) 43 . Protein tagging, gene deletion, and mutant alleles were generated at the endogenous genomic loci following standard PCR-based or CRISPR–Cas9 based methods 29 . All genetically altered loci were verified by sequencing. Standard yeast genetic manipulation was used for tetrad analyses and all experiments were conducted at 30ºC unless noted. At least two different biological isolates per genotype were used for each assay. Genotoxin sensitivity tests. Spotting assays to detect DNA damage sensitivity were carried out as described previously 44 . In brief, yeast cultures were grown overnight at 30°C in rich medium (YPD). Cultures were then diluted to OD 600 0.15 and allowed to grow to early mid-log phase in YPD. Subsequently, cells were spotted in 10-fold serial dilutions on plates containing YPD with or without DNA damaging agents at the indicated concentrations. Plates were incubated at 30°C and photographed after 2–4 days. Synchronization and cell cycle analysis. The cell synchronization procedure was used as previously described with a few modifications 45 . In brief, early-mid log-phase yeast cultures grown in the YPD media at OD 600 ~ 0.2 were treated with α factor for G1 arrest. After the initial addition of 5 µg/ml α factor (Thermo Fisher) for one hour, a second dose of 2.5 µg/ml α factor was added for another hour. Cell morphology was monitored to confirm G1 arrest. Subsequently, cells were released from G1 arrest by growing in YPD media containing 100 µg/ml protease (Sigma) to degrade α factor. The media also contained 0.03% MMS for genotoxin treatment. After 45 min, MMS was washed out, and cells were allowed to grow in YPD media. Samples were taken every 20 min for FACS analyses and Rad53 examination. FACS analyses were done as previously described 45 . Briefly, cells were fixed in 70% ethanol for 1 h at room temperature or overnight at 4°C. RNAs and proteins were degraded by sequential treatment with RNase A (Sigma) and Protease K (Sigma). DNA was stained with Sytox Green (Invitrogen). Samples were examined using a BD LSR II flow cytometer (BD). Data analyses were performed using the FlowJo Software (BD). Protein extraction to examine Rad53 phosphorylation. Protein extracts were made using a TCA (Trichloroacetic acid) method to maintain Rad53 phosphorylation as described previously 46 . In brief, cells were homogenized using 0.5 mm silica beads (BioSpec Products) in the presence of 20% TCA on a FastPrep-24 bead-beating grinder (MP Biomedicals). After removing the supernatant by centrifugation, the precipitated proteins were washed with 5% cold TCA, and then dissolved in protein loading buffer (50 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.05% bromophenol blue). Samples were boiled for 5 min before being loaded onto 4–20% SDS-PAGE gels (Bio-Rad) and subsequently examined by immunoblotting (see below). Co-immunoprecipitation. Standard protocols were followed to perform co-immunoprecipitation. In brief, cells were disrupted by bead beating in lysis buffer that contained 20 mM HEPES-KOH (pH 7.5), 100 mM KOAc, 1% Triton X-100, 2 mM Mg(OAc) 2 , 1 mM NaF, 2 mM β-glycerophosphate, and EDTA-free protease inhibitors (Roche). Lysates were cleaned by centrifugation at 20,000g for 30 min to obtain whole-cell extract, which was then incubated with anti-Flag beads (Sigma-Aldrich) or anti-HA beads (Pierce) for 2 h at 4°C. After washing the beads three times with lysis buffer, bead-bound proteins were eluted using loading buffer as described above. Proteins were boiled for 5 min before being loaded onto 4–20% gradient gels (Bio-Rad) for electrophoresis and immunoblotting (see below). Immunoblotting and antibodies used. Proteins were transferred from gels to 0.2 µm nitrocellulose membranes (GE) before immunoblotting. The antibodies used include: anti-phosphorylated active Rad53 (F9, a gift from Marco Foiani and Daniele Piccini), anti-Ddc1 (a gift from Marco Muzi-Falconi), anti-Dpb11 (a gift from Dirk Remus), anti-Rad9 (a gift from John Petrini), anti-Histone H3 (ab1791, Abcam), anti-Actin (C4, 8691001, MP Biomedicals), anti-Sir2 (yN-19, sc-6666, Santa Cruz), anti-Flag (M2, F1804, Sigma), anti-HA (F-7, sc-7392, Santa Cruz), anti-Myc (9E10, BE0238, Bio X Cell), anti-TAP (PAP, P1291, Sigma), anti-Pgk1 (22C5, Molecular Probes), and anti-Tubulin (YL1/2, ab6160, Abcam), Chromatin fractionation. Chromatin fractionation was performed as described previously with a few modifications 47 . In brief, yeast spheroplasts derived from log-phase cells were lysed using extraction buffer (150 mM KOAc, 20 mM pH 6.6 PIPES-KOH, 2 mM Mg(OAc) 2 , 1 mM NaF, 0.5 mM Na 3 VO 4 , 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) by incubating on ice for 5 min in the presence Zymolyase. Lysates were then subjected to centrifugation at 16,000 g for 15 min at 4°C on a sucrose cushion. The resultant chromatin pellets were washed and resuspended with extraction buffer. Loading buffer was added to each sample before boiling for 5 min. Samples were then examined by SDS-PAGE followed by immunoblotting. Tubulin or Pgk1 was used as a marker of the non-chromatin fraction, while histone H3 or Sir2 was used as a marker for chromatin-associated proteins. Protein stability assay. Protein stability was examined using a standard protocol as described previously 48 , 49 . Briefly, yeast cultures were grown to OD 600 0.2 before CHX was added to a final concentration of 100 µg/ml to inhibit protein synthesis. Equal numbers of cells were collected every 1 h following CHX addition. Cell lysates were examined using the TCA method described above. The band intensity of each protein after immunoblotting was quantified using the FIJI software 50 . rDNA marker loss and GCR assays . Standard protocols were followed for both assays. For the GCR assay that assesses the loss of the URA3 and CAN1 markers inserted at the YEL068c locus, at least six cultures were examined for each genotype 29 , 51 . Cells were plated on SC (synthetic complete) media containing 5-FOA to counter-select URA3 and containing canavanine to counter-select CAN1 . The numbers of colonies grown on 5-FOA- containing SC (FC) plates reflect those that lost URA3 and CAN1 markers. Cells were also plated on SC plates to determine the total viable colonies. GCR rates were calculated as m/N T by the following formula: m[1.24 + ln(m)] = N FC . Here, m represents mutational events, N FC is the number of colonies on FC plates, and N T is the number of colonies on SC plates. The upper and lower 95% confidence intervals were derived. Frequencies for the loss of the ADE2 and CAN1 markers inserted into the rDNA array were measured as described previously 52 . Cells were grown to stationary phase over equal doubling times and plated on SC media to count total viable colonies. Cells were also plated on media containing canavanine (SC + Can) to select those that lost the CAN1 marker. The frequency of marker loss was calculated as: F = N can /N C , where N Can is the number of colonies on SC + Can plates and N C is the number of cells plated on SC plates 53 . Declarations AUTHOR CONTRIBUTION: All authors were involved in research design and data analyses. B.W., D.G., S.L, and T.C.E performed experiments. B.W. and X.Z. wrote the paper with all the authors’ input. ACKNOWLEDGEMENTS: We thank Drs. Marco Foiani, Daniele Piccini, Marco Muzi-Falconi, Dirk Remus and John Petrini for sharing antibodies. B. Wan. is supported by NSFC grant 32170087. X.Z. is supported by National Institute of General Medical Sciences (NIGMS) grants R35GM145260. T.C.E is supported by a Kravis WiSE Fellowship. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. DECLARATION OF INTERESTS. The authors declare no competing interests. References Hartwell LH, Weinert TA (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246:629–634 Enoch T, Carr AM, Nurse P (1992) Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev 6:2035–2046 Lanz MC, Dibitetto D, Smolka MB (2019) DNA damage kinase signaling: checkpoint and repair at 30 years. EMBO J 38:e101801 Usui T, Ogawa H, Petrini JH (2001) A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol Cell 7:1255–1266 Zou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300:1542–1548 Elledge SJ (1996) Cell cycle checkpoints: preventing an identity crisis. Science 274:1664–1672 Foiani M et al (2000) DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae. Mutat Res 451:187–196 Harrison JC, Haber JE (2006) Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40:209–235 Faca VM et al (2020) Maximized quantitative phosphoproteomics allows high confidence dissection of the DNA damage signaling network. Sci Rep 10:18056 Nyberg KA, Michelson RJ, Putnam CW, Weinert TA (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 36:617–656 Clemenson C, Marsolier-Kergoat MC (2009) DNA damage checkpoint inactivation: adaptation and recovery. DNA Repair (Amst) 8:1101–1109 Waterman DP, Haber JE, Smolka MB (2020) Checkpoint responses to DNA double-strand breaks. Annu Rev Biochem 89:103–133 Pizzul P et al (2022) The DNA damage checkpoint: A tale from budding yeast. Front Genet 13:995163 Ohouo PY, Bastos de Oliveira FM, Liu Y, Ma CJ, Smolka MB (2013) DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9. Nature 493:120–124 Gobbini E et al (2015) Sae2 function at DNA double-strand breaks Is bypassed by dampening Tel1 or Rad53 activity. PLoS Genet 11:e1005685 Yu TY, Kimble MT, Symington LS (2018) Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection. Proc. Natl. Acad. Sci. U. S. A. 115, E11961-E11969 Dhingra N et al (2021) The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin. Proc. Natl. Acad. Sci. U. S. A. 118 Pizzul P et al (2024) Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding. Nucleic Acids Res 52:2355–2371 Wysocki R et al (2005) Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9. Mol Cell Biol 25:8430–8443 Hammet A, Magill C, Heierhorst J, Jackson SP (2007) Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast. EMBO Rep 8:851–857 Vialard JE, Gilbert CS, Green CM, Lowndes NF (1998) The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J 17:5679–5688 Sanchez Y et al (1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271:357–360 Ohouo PY, Bastos de Oliveira FM, Almeida BS, Smolka MB (2010) DNA damage signaling recruits the Rtt107-Slx4 scaffolds via Dpb11 to mediate replication stress response. Mol Cell 39:300–306 Chin JK, Bashkirov VI, Heyer WD, Romesberg FE (2006) Esc4/Rtt107 and the control of recombination during replication. DNA Repair (Amst) 5:618–628 Mimura S et al (2010) Cul8/Rtt101 forms a variety of protein complexes that regulate DNA damage response and transcriptional silencing. J Biol Chem 285:9858–9867 Hang LE et al (2015) Rtt107 is a multi-functional scaffold supporting replication progression with partner SUMO and ubiquitin ligases. Mol Cell 60:268–279 Zaidi IW et al (2008) Rtt101 and Mms1 in budding yeast form a CUL4(DDB1)-like ubiquitin ligase that promotes replication through damaged DNA. EMBO Rep 9:1034–1040 Wan B, Hang LE, Zhao X (2016) Multi-BRCT scaffolds use distinct strategies to support genome maintenance. Cell cycle (Georgetown Tex) 15:2561–2570 Wan B, Wu J, Meng X, Lei M, Zhao X (2019) Molecular basis for control of diverse genome stability factors by the multi-BRCT scaffold Rtt107. Mol. Cell 75, 238–251 e235 Pfander B, Matos J (2017) Control of Mus81 nuclease during the cell cycle. FEBS Lett 591:2048–2056 Bermejo R et al (2007) Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev 21:1921–1936 Puddu F et al (2008) Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint. Mol Cell Biol 28:4782–4793 Osborn AJ, Elledge SJ (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev 17:1755–1767 Paciotti V, Clerici M, Scotti M, Lucchini G, Longhese MP (2001) Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway. Mol Cell Biol 21:3913–3925 Emili A (1998) MEC1-dependent phosphorylation of Rad9p in response to DNA damage. Mol Cell 2:183–189 Li X et al (2012) Structure of C-terminal tandem BRCT repeats of Rtt107 protein reveals critical role in interaction with phosphorylated histone H2A during DNA damage repair. J Biol Chem 287:9137–9146 Balint A et al (2015) Assembly of Slx4 signaling complexes behind DNA replication forks. EMBO J 34:2182–2197 Bonner JN et al (2016) Smc5/6 mediated sumoylation of the Sgs1-Top3-Rmi1 complex promotes removal of recombination intermediates. Cell Rep 16:368–378 Tsabar M et al (2016) Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair. Genes Dev 30:1211–1224 Millan-Zambrano G et al (2018) Phosphorylation of histone H4T80 triggers DNA damage checkpoint recovery. Mol Cell 72:625–635e624 Cussiol JR, Dibitetto D, Pellicioli A, Smolka MB (2017) Slx4 scaffolding in homologous recombination and checkpoint control: lessons from yeast. Chromosoma 126:45–58 Lanz MC et al (2018) Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling. Genes Dev 32:822–835 Zhao X, Blobel G (2005) A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc. Natl. Acad. Sci. U. S. A. 102, 4777–4782 Meng X et al (2020) DNA polymerase epsilon relies on a unique domain for efficient replisome assembly and strand synthesis. Nat Commun 11:2437 Wei L, Zhao X (2016) A new MCM modification cycle regulates DNA replication initiation. Nat Struct Mol Biol 23:209–216 Li S et al (2021) Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance. Genes Dev 35:261–272 Chung I, Zhao X (2015) DNA break-induced sumoylation is enabled by collaboration between a SUMO ligase and the ssDNA-binding complex RPA. Genes Dev 29:1593–1598 Belle A, Tanay A, Bitincka L, Shamir R, O'Shea EK (2006) Quantification of protein half-lives in the budding yeast proteome. Proc. Natl. Acad. Sci. U. S. A. 103, 13004–13009 Han J et al (2010) Ubiquitylation of FACT by the cullin-E3 ligase Rtt101 connects FACT to DNA replication. Genes Dev 24:1485–1490 Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682 Putnam CD, Kolodner RD (2010) Determination of gross chromosomal rearrangement rates. Cold Spring Harb Protoc pdb prot5492 (2010) Fritze CE, Verschueren K, Strich R (1997) Easton Esposito, R. Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA. EMBO J 16:6495–6509 Regan-Mochrie G et al (2022) Yeast ORC sumoylation status fine-tunes origin licensing. Genes Dev 36:807–821 Zhao X, Muller EG, Rothstein R (1998) A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell 2:329–340 Additional Declarations There is NO Competing Interest. Supplementary Files nrreportingsummaryMms22.pdf Reporting Summary Mms22SupplFig.pdf Supp.Table.docx Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4417144","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":306101448,"identity":"e6b935d4-6a4a-4fbc-9360-a6636b8bf393","order_by":0,"name":"Xiaolan Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYDACCRBhYMPAwEOaloI0krV8OEyCFvnZzQ8f8xicj5bvOfzwA0PFPbsGQloY5xwzNpxhcDt3w9k2YwmGM8XJBLUwSySYSXwAaeHnYWNgbEtIJugwNon0bxIJBudy5/cTq4VHIgdky4HchrM9YC12BLVISOQUA/2SnLvhzDFjiYQzCQkEtcjPSN/4mOePXe78nuSHHz5UJNgT1IIKgFYkNpCoh4GBVFtGwSgYBaNgBAAAD3E5YvlslzwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8302-6905","institution":"Memorial Sloan Kettering Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Xiaolan","middleName":"","lastName":"Zhao","suffix":""},{"id":306101449,"identity":"aeae0a18-6784-40d5-974c-2edc230ff22e","order_by":1,"name":"Bingbing Wan","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Bingbing","middleName":"","lastName":"Wan","suffix":""},{"id":306101450,"identity":"2729d776-ee5f-4564-80a6-e9f6f1d3981e","order_by":2,"name":"Danying Guan","email":"","orcid":"","institution":"Memorial Sloan Kettering Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Danying","middleName":"","lastName":"Guan","suffix":""},{"id":306101451,"identity":"c964da55-7d5f-4a46-be79-d0dba93bd7cb","order_by":3,"name":"Shibai Li","email":"","orcid":"","institution":"Memorial Sloan Kettering Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Shibai","middleName":"","lastName":"Li","suffix":""},{"id":306101452,"identity":"92ffb37a-46b2-48ef-a208-793141a4e930","order_by":4,"name":"Tzippora Chwat-Edelstein","email":"","orcid":"","institution":"Memorial Sloan Kettering Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Tzippora","middleName":"","lastName":"Chwat-Edelstein","suffix":""}],"badges":[],"createdAt":"2024-05-14 07:15:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4417144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4417144/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-54624-0","type":"published","date":"2025-01-02T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57065280,"identity":"396796ec-2acd-4131-a962-52fad75b9b2e","added_by":"auto","created_at":"2024-05-24 07:05:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":665907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisrupting Mms22 binding to Rtt107 leads to genotoxin sensitivity and persistent DNA damage checkpoint.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic showing that Mms22 uses its RIM sequence to bind to the N-terminal (N-ter) tetra-BRCT domain of the scaffold Rtt107 protein, while gH2A binds to the C-terminal (C-ter) domain of Rtt107\u003csup\u003e29, 36\u003c/sup\u003e. The \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e mutant specifically disrupts Mms22 binding to Rtt107\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cem\u003e mms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003emms22∆\u003c/em\u003e cells exhibited sensitivity to three genotoxins. Cells were spotted in 10-fold serial dilutions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells are defective in exiting the G2/M phase compared with wild-type cells after transient exposure to MMS. The experimental scheme is depicted on the right. Represented FACS profiles are shown on the left.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e The active form of Rad53 persists in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e but not in wild-type cells after transient exposure to MMS. Samples were taken in the same experiments as those in panel \u003cstrong\u003ec\u003c/strong\u003e. The active form of Rad53 was detected by the F9 antibody. β-Actin served as the loading control.\u003c/p\u003e","description":"","filename":"Mms22Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/32ea41c90953b088a093443f.png"},{"id":57065281,"identity":"fcbd498d-d6a3-4143-920e-e73e3224836b","added_by":"auto","created_at":"2024-05-24 07:05:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1282947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThree related phenotypes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emms22\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eRIM\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e cells are rescued by mild mutant alleles of the DNA damage checkpoint proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eMMS sensitivity of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells is suppressed by point mutations in the DNA damage checkpoint protein Rad9 or Ddc1. Experiments were done as in Fig. 1b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e The MMS sensitivity of\u003cem\u003e mms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells is not affected by mutations reducing the DNA replication checkpoint, including \u003cem\u003emrc1-AQ\u003c/em\u003e and \u003cem\u003emec1-100\u003c/em\u003e. Experiments were done as in Fig. 1b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Defects in exiting the G2/M phase seen in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells are improved by mutating Rad9. FACS profiles of indicated strains are shown. Arrows highlight different amounts of cells that have exited the first G2/M phase and entered the second G1 phase at 300 min for two of the examined strains. Experiments were done as in Fig. 1c.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Percentages of cells that have exited the G2/M phase and entered the next G1 phase. The calculation was based on three biological duplicates for the indicated time points. Averages and Standard Error of the Means (SEMs) are indicated; statistical analysis was performed by one-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Persistent Rad53 activation in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells is suppressed by \u003cem\u003erad9-K1088M. \u003c/em\u003eExperiments were done as in Fig. 1d, and α-Tubulin served as the loading control.\u003c/p\u003e","description":"","filename":"Mms22Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/6f113df0ec1db6dde356761a.png"},{"id":57064798,"identity":"b41db355-dc3d-4c1d-8a78-fcfe99d9deb2","added_by":"auto","created_at":"2024-05-24 06:57:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":662568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Mms22-Rtt107 axis acts in parallel to the Slx4-Rtt107 axis in checkpoint dampening.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003e\u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e are additive in delaying the exit from the G2/M phase after transient exposure to MMS. Experiments were done as in Fig. 1c. Left: FACS profiles of indicated strains, with arrows highlighting different amounts of G1 cells in the mutants toward the end of the time course. Right: percentages of cells that have exited the G2/M phase at the indicated time point were calculated based on three biological duplicates. Averages and SEMs are indicated; statistical analysis was performed by one-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e show additive effect for increasing the levels of active Rad53. Experiments were done as in Fig. 1d. Top: representative immunoblotting results detecting Rad53 phosphorylation at indicated time points. Tubulin served as the loading control. Bottom: relative levels of phosphorylated Rad53 calculated based on three biological duplicates. Averages and SEMs are indicated; statistical analysis was determined by one-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e are additive in causing MMS sensitivity. Experiments were done as in Fig. 1b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e The effects of\u003cem\u003e mms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e on the chromatin association of Mms22 and Slx4. Sir2 and Tubulin were used to mark chromatin-bound and non-chromatin-bound fractions, respectively. Left: cells contained Flag-tagged Mms22 and Slx4. Right: cells contained HA tagged Mms22 and TAP-tagged Slx4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e A model to summarize the Mms22-Rtt107 and Slx4-Rtt107 pathways in checkpoint dampening as suggested by our data presented in Figs. 1-3.\u003c/p\u003e","description":"","filename":"Mms22Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/31629601ba4249caf9553439.png"},{"id":57065282,"identity":"a531f60e-d6fa-45a7-bb31-28dbb57c0fb3","added_by":"auto","created_at":"2024-05-24 07:05:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":692998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMms22, Rtt107, and the Rtt101\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eMms1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e E3 contribute to Rad9 instability.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eRad9 protein stability examined in the presence of cycloheximide (CHX) that blocks new protein synthesis. Both wild-type and indicated mutants were examined during growth. Actin served as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Quantification of Rad9 protein stability during normal growth. Rad9 protein levels examined during the time course as exemplified in panel A were quantified in reference to the loading control of actin levels. Averages and SEMs are shown based on at least two biological duplicates; statistical analysis for each time point was performed by one-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e-test. * p values \u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Rad9 protein stability was examined when cells were treated with MMS and CHX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Quantification of Rad9 protein stability in MMS-treated conditions. Experiments were done as panel A and data are presented as panel B except that cells were treated with MMS during the time course. Averages and SEMs are shown based on at least two biological duplicates; statistical analysis for each time point was determined by one-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e-test. * p values \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Mms22Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/8af1f944c5c3efb1765ed9a0.png"},{"id":57064800,"identity":"a3694ab3-4b2e-41b9-8bdf-69d46479835f","added_by":"auto","created_at":"2024-05-24 06:57:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":756412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRad9 degradation is proteasome-dependent and a \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003erad9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant rescues the MMS sensitivity of the Rtt101\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eMMS1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e E3 mutants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eExamination of\u003cstrong\u003e \u003c/strong\u003eRad9 protein stability in the presence of MG132 that blocks proteasomal functions or DMSO control. Cells were simultaneously treated with cycloheximide (CHX) to block new protein synthesis. Left, a representative immunoblotting result showing that Rad9 was better stabilized in the presence of MG132 compared with the DMSO treatment. Right: quantification of Rad9 protein levels during the time courses in reference to the loading control of actin levels from three biological duplicates. Averages and SEMs are shown based on at least three biological duplicates; statistical analysis for each timepoint was performed by one-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e-test. * p \u0026lt;0.05, ** p\u0026lt; 0.01, *** p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Sensitivity of \u003cem\u003ertt101∆\u003c/em\u003e and \u003cem\u003emms1∆\u003c/em\u003e cells toward three genotoxins is rescued by reducing Rad9 function via the \u003cem\u003erad9-K1088M\u003c/em\u003e mutation. Experiments were done as in Fig. 1b.\u003c/p\u003e","description":"","filename":"Mms22Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/0c35dc2b0b6b10e1109a0320.png"},{"id":57064802,"identity":"98a230c2-dcdc-4e0f-a8d6-c50d2753f1f2","added_by":"auto","created_at":"2024-05-24 06:57:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":518779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Mms22-Rtt107 interaction promotes chromatin association of the Rtt101\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eMMS1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e E3 and degradation of chromatin associated Rad9.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells show increased level of Rad9 on chromatin. Histone H3 and β-Actin mark chromatin-bound and non-chromatin-bound protein samples, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Disrupting the Mms22 and Rtt107 binding via \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e reduces the chromatin association of Mms1 and Rtt101. Histone H3 or Sir2 marks chromatin-bound samples, while and Pgk1 marks non-chromatin-bound protein samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec \u003c/strong\u003e\u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e leads to the stabilization of Rad9 on chromatin. Degradation of Rad9 in chromatin fraction and whole cell extract (WCE) was examined in the presence of CHX that blocks protein synthesis. Sir2 and Actin mark chromatin-bound and non-chromatin-bound protein samples, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed \u003c/strong\u003eA\u003cstrong\u003e \u003c/strong\u003emodel suggesting that Rtt107 binding to nucleosome markers such as γH2A helps to recruit Mms22-containing Rtt101\u003csup\u003eMms1\u003c/sup\u003e ubiquitin E3 to chromatin that can facilitate Rad9 degradation. This mechanism acts in parallel with the Slx4-Rtt107 mediated displacement of Rad9 from chromatin. The combined effects of these two Rtt107-mediated pathways can provide more potent Rad9 silencing than either pathway alone in reducing the DNA damage checkpoint signaling. P: phosphorylation on γH2A; M: methylated H3K79\u003c/p\u003e","description":"","filename":"Mms22Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/d43324eeb2693b7551a281cf.png"},{"id":72949916,"identity":"e2d72e0e-a933-42a8-a0fe-22bdbda4c407","added_by":"auto","created_at":"2025-01-04 08:16:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5563423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/4f7746aa-5339-48e4-a716-22aadf85049d.pdf"},{"id":57064793,"identity":"955523b7-7ed1-4b1c-bb53-3f26e1786423","added_by":"auto","created_at":"2024-05-24 06:57:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":87368,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"nrreportingsummaryMms22.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/cba258015861565a32a3910e.pdf"},{"id":57064795,"identity":"06f97c97-9485-4f87-9a06-46d9376a128d","added_by":"auto","created_at":"2024-05-24 06:57:12","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":774587,"visible":true,"origin":"","legend":"","description":"","filename":"Mms22SupplFig.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/1267cb27a7b92c441adad9fc.pdf"},{"id":57064797,"identity":"f5433b7f-78f2-4021-86f3-c2465ef7e906","added_by":"auto","created_at":"2024-05-24 06:57:12","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":43156,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-4417144/v1/baede6089e87cc44ec04c773.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The Mms22-Rtt107 axis dampens the DNA damage checkpoint by reducing the stability of the Rad9 checkpoint mediator","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe highly conserved DNA damage checkpoint (DDC) is a critical component of the genome stress response\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. When cells suffer from increased burdens of genome lesions caused by genotoxins or endogenous sources, the DNA damage sensor proteins can recruit apical DDC kinases to DNA lesion sites leading to their subsequent activation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. DDC mediator proteins can then amplify the genome stress signals by recruiting downstream transducer kinases, which can be phosphorylated and activated by the apical DDC kinases\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The activated transducer kinases can diffuse to various cellular compartments and phosphorylate hundreds of substrates\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Such a large-scale phosphoproteomic transformation can induce a myriad of cellular changes. These include cell cycle arrest to provide more time for DNA repair, increased ability to repair, and transcriptional alterations, among many other consequences\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While DDC-induced changes are beneficial for cells to cope with stress temporarily, long-term survival also depends on the ability to dampen the checkpoint once stress is dealt with or when stress becomes persistent\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Checkpoint dampening can permit cell cycle resumption and thus the chance of continued growth, as well as access to DNA repair mechanisms operating in different cell cycle phases, among other benefits\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, the various strategies employed to dampen DDC remain to be discovered.\u003c/p\u003e \u003cp\u003eDDC dampening has thus far been mostly examined in the model organism budding yeast. Initial studies have focused on protein phosphatases that can reverse some of the phosphorylation changes elicited by the DDC kinases\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, phosphatases alone are insufficient for DDC dampening, and cells also need to reduce the continuous emission of DDC signals generated through the chains of the DNA damage sensors, apical DDC kinases, checkpoint mediators, and transducer kinases. Examples of phosphatase-independent strategies to downregulate DDC include displacing checkpoint factors from chromatin\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Whether and how additional pathways can reduce DDC signaling remain to be elucidated.\u003c/p\u003e \u003cp\u003eHere we report a strategy of DDC dampening mediated by proteasomal degradation of a key checkpoint mediator protein Rad9 in yeast. Rad9 associates with DNA lesion sites in response to genotoxin treatment through binding to nucleosomes containing γH2A that demarcate damage domains and methylated H3K79\u003csup\u003e19, 20\u003c/sup\u003e. Rad9 can then recruit the transducer kinase Rad53 in preparation for Rad53 phosphorylation and subsequent activation by the main apical DDC kinase, Mec1\u003csup\u003e21, 22\u003c/sup\u003e. As Rad53 is responsible for a large majority of DDC-induced phosphorylation events in yeast, regulating its upstream enabler, Rad9, can be a key step to downregulate DDC.\u003c/p\u003e \u003cp\u003eIndeed, a scaffolding complex composed of the Rtt107 and Slx4 proteins was shown to compete with Rad9 for binding to nucleosomes, thus reducing Rad9\u0026rsquo;s association with DNA lesion sites\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Interestingly, Rtt107 can additionally bind to another scaffold protein Mms22\u003csup\u003e24\u0026ndash;26\u003c/sup\u003e. Mms22 was shown to serve as a substrate adaptor of the cullin ubiquitin E3 complex, which also contains the E3 subunit Rtt101 and the Mms1 subunit (Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. While the Mms22-Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 complex is known to protect genome stability and support cell growth when facing genotoxins, its functional mechanisms are not fully understood\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We show here that Rtt107 binding to Mms22 as well as the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 are required for Rad9 degradation. We also demonstrate that Mms22 and Slx4 act in parallel to achieve a more effective \u0026ldquo;anti-Rad9\u0026rdquo; outcome during DDC recovery. As such, Rtt107 enables a two-pronged strategy to downregulate Rad9-mediated DDC via collaborating with its two binding partners.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eRtt107 association with Mms22 prevents genotoxin sensitivity and prolonged checkpoint\u003c/h2\u003e \u003cp\u003eBoth Rtt107 and Mms22 are important for genome stability; however, the biological functions of their interaction have been unclear. To address this question, we examined a separation-of-function allele of Mms22 that specifically disrupts the Rtt107 binding without affecting other known interactions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Given that Rtt107 and Mms22 each have multiple binding partners, this allele provides a valuable reagent to define the biological functions of their interaction\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. High-resolution structure has revealed that Rtt107 binds to the N-terminal Rtt107-interaction-motif (RIM) of Mms22\u003csup\u003e29\u003c/sup\u003e. Further, alanine substitution of two residues (D13 and Y33) within Mms22\u0026rsquo;s RIM (\u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e) disrupts the Mms22 and Rtt107 interaction (Fig.\u0026nbsp;1a)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e supports the wild-type level of protein expression and interactions with other binding partners, such as the Mms1 protein that associates with the C-terminal region of Mms22 (Supplementary Fig.\u0026nbsp;1a)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We confirmed here that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e led to sensitivity to the DNA methylation agent MMS (methyl methanesulfonate) and further showed that it also caused sensitivity to the Top1 trapping compound CPT (camptothecin) and the replication fork blocking agent HU (hydroxyurea) (Fig.\u0026nbsp;1b). Compared with \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003emms22∆\u003c/em\u003e led to greater sensitivity to MMS, CPT, and HU, as well as slower growth (Fig.\u0026nbsp;1b). This agrees with the notion that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e is a separation-of-function allele affecting the Mms22-Rtt107 binding without interfering with other roles of Mms22.\u003c/p\u003e \u003cp\u003eSensitivity to several genotoxins raised the possibility that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e may affect common processes during different genotoxin responses such as the DNA damage checkpoint. To test this notion and determine the underlying causes of the genotoxin sensitivity exhibited by \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells, we focused on MMS treatment since DDC has been well examined in this condition. Specifically, we examined how synchronized cells moved through the cell cycle in a regimen that allows monitoring of both the initial response to MMS and the recovery. Briefly, cells synchronized in G1 were released to the cell cycle in the presence of MMS for 45 minutes, and then allowed to recover in MMS-free media (Fig.\u0026nbsp;1c, right). As expected, wild-type cells progressed slowly through S phase and were able to enter the second cell cycle after MMS washout (Fig.\u0026nbsp;1c). We also examined Rad53 activation, a marker for DDC signaling. The F9 antibody is widely used to detect Rad53 activation as it specifically recognizes the phosphorylated but not unmodified form of Rad53, although the antibody also binds to a nonspecific band only in G1 cells\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. As previously reported, we observed that in wild-type cells, Rad53 activation was induced in S phase when cells were treated with MMS (30\u0026ndash;45 min) and gradually diminished during the recovery phase (Fig.\u0026nbsp;1d and Supplementary Fig.\u0026nbsp;1b).\u003c/p\u003e \u003cp\u003eIn contrast to wild-type cells, \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells delayed the re-entry into the second G1 phase after MMS washout, though showed no obvious defects during S phase progression in the presence of MMS (Fig.\u0026nbsp;1c). Concomitantly, Rad53 phosphorylation induced by MMS failed to decline during the recovery phase (Fig.\u0026nbsp;1d and Supplementary Fig.\u0026nbsp;1b). The results from these two assays are consistent with each other and suggest that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells exhibit persistent DDC during the recovery phase, which can delay entry to the next cell cycle.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenotoxin sensitivity associated with the loss of the Mms22-Rtt107 interaction is rescued by reducing the DNA damage checkpoint\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePersistent checkpoint can be caused by delayed genome replication and repair or by reduced ability to dampen the checkpoint itself. The two scenarios differ in how cells would respond to reduced levels of DDC. In the first scenario, cell viability would suffer when checkpoint function is weakened, because cells rely on optimal checkpoint to complete DNA replication and repair. In contrast, cells in the second scenario could show better survival upon checkpoint weakening. Based on this rationale, we tested how \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells responded to reduced DDC levels conferred by mild alleles of the checkpoint mediator protein Rad9 or Ddc1. The two mutants used, namely \u003cem\u003erad9-K1088M\u003c/em\u003e and \u003cem\u003eddc1-T602A\u003c/em\u003e, contain single point mutations that reduce Rad9 binding to γH2A and Ddc1 binding to another checkpoint factor Dpb11\u003csup\u003e20, 32\u003c/sup\u003e. These studies have shown that \u003cem\u003erad9-K1088M\u003c/em\u003e and \u003cem\u003eddc1-T602A\u003c/em\u003e mildly reduce DDC. Significantly, we found that either allele conferred strong rescue of the MMS sensitivity of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells (Fig.\u0026nbsp;2a).\u003c/p\u003e \u003cp\u003eWhile the DNA damage checkpoint can operate throughout the cell cycle, cells also employ the DNA replication checkpoint (DRC) during S phase\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. We next examined whether mild hypomorphic DRC mutants could affect the MMS sensitivity of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells. We tested two well-characterized alleles affecting either the Mrc1 mediator protein of the DRC pathway, namely \u003cem\u003emrc1-AQ\u003c/em\u003e, or \u003cem\u003emec1-100\u003c/em\u003e that is specifically defective in DRC\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;2b, neither allele affected the MMS sensitivity of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells. Together, these data suggest that defects of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells can be rescued by reducing DDC, but not DRC, functions. Collectively, the genetic findings raise the possibility that Mms22 binding to Rtt107 contributes to the downregulation of DDC but not DRC, and that this role can be partly responsible for the genotoxin sensitivity caused by the loss of the Mms22-Rtt107 interaction.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePersistent checkpoint in\u003c/b\u003e \u003cb\u003emms22-RIM\u003c/b\u003e \u003cb\u003ecells is rescued by\u003c/b\u003e \u003cb\u003erad9-K1088M\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eddc1-T602A\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further test the above hypothesis, we examined whether the suppression of MMS sensitivity of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells by the DDC mutants is associated with a correction of the persistent checkpoint seen in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells. We used a similar experimental scheme as depicted in Fig.\u0026nbsp;1c to monitor cell cycle progression and Rad53 activation, except a longer period of recovery was examined. Similar to observations described above (Fig.\u0026nbsp;1c), while wild-type cells were able to enter the next cell cycle after recovery from MMS treatment, \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e delayed the entry (Fig.\u0026nbsp;2c and Supplementary Fig.\u0026nbsp;2a). This delay was quantified for two late time points (260 and 300 min; Fig.\u0026nbsp;2d). Significantly, this delay was improved by either \u003cem\u003erad9-K1088M\u003c/em\u003e or \u003cem\u003eddc1-T602A\u003c/em\u003e (Fig.\u0026nbsp;2c and Supplementary Fig.\u0026nbsp;2a). Compared with \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e, more cells in the \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e \u003cem\u003erad9-K1088M\u003c/em\u003e or \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eddc1-T602A\u003c/em\u003e double mutants exited G2/M and entered the next G1 phase between 200 to 300 minutes, with the most prominent differences seen at 260 and 300 minutes (Fig.\u0026nbsp;2d). \u003cem\u003erad9-K1088M\u003c/em\u003e and \u003cem\u003eddc1-T602A\u003c/em\u003e behaved similarly to wild-type cells, reflecting redundancy among DDC mediator functions (Figs.\u0026nbsp;2c and 2d, Supplementary Fig.\u0026nbsp;2a). Importantly, \u003cem\u003erad9-K1088M\u003c/em\u003e or \u003cem\u003eddc1-T602A\u003c/em\u003e also reduced the persistent Rad53 activation seen in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells (Fig.\u0026nbsp;2e and Supplementary Fig.\u0026nbsp;2b). Collectively, the suppression of prolonged Rad53 activation and cell cycle arrest seen in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e by \u003cem\u003erad9-K1088M\u003c/em\u003e or \u003cem\u003eddc1-T602A\u003c/em\u003e provides further evidence that Mms22 binding to Rtt107 plays a role in dampening the DNA damage checkpoint.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMms22 and Slx4 act in parallel to dampen the DNA damage checkpoint\u003c/h2\u003e \u003cp\u003eRtt107 has an established role in DDC dampening through pairing with Slx4\u003csup\u003e14, 23\u003c/sup\u003e. Rtt107 employs the same surface to engage with short motifs within Slx4 or Mms22, engendering mutually exclusive pairwise interactions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. A separation-of-function allele, \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e (T423, T424, and S567A), has been constructed that specifically abolishes the Slx4-Rtt107 interaction without affecting protein level or binding to other known partners, such as the Slx1 nuclease\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We thus asked how the Mms22-Rtt107 mediated effect on DDC recovery is functionally related to that mediated by Slx4-Rtt107.\u003c/p\u003e \u003cp\u003eApplying the experimental scheme described in Fig.\u0026nbsp;2A, we found that \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e led to a more pronounced delay in late S phase compared with \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e (80 and 120 min; Fig.\u0026nbsp;3a). In contrast, \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e showed a milder delay in re-entry into the next cell cycle compared with \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e (240 and 300 min; Fig.\u0026nbsp;3a). These observations raised the possibility that the two Rtt107 interactors may differentially affect DDC at different stages of the cell cycle.\u003c/p\u003e \u003cp\u003eSignificantly, the \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e double mutant exhibited more severe defects in exiting the G2/M arrest in the first cell cycle than either single mutant (Fig.\u0026nbsp;3a). At the end of the time course (300 min), the double mutant showed significantly fewer cells exited the G2/M phase than either single mutant (Fig.\u0026nbsp;3a). A similar additive effect was seen when assaying for the active form of Rad53. The \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e double mutant showed higher levels of active Rad53 than either single mutant at the two last time points of the time course (240 and 300 min; Fig.\u0026nbsp;3b). Results from cell cycle analysis and active Rad53 forms can be best explained by that Mms22-Rtt107 and Slx4-Rtt107 complexes acting in different pathways to downregulate the DNA damage checkpoint.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAdditional evidence supports the independence of Mms22- and Slx4-mediated functions\u003c/h3\u003e\n\u003cp\u003eFurther supporting the functional independence of Mms22 and Slx4, we found that the double mutant of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e led to stronger MMS sensitivity than either single mutant (Fig.\u0026nbsp;3c). The additive effect of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e was also seen when assaying the stability of the repetitive ribosomal DNA (rDNA) locus and of a non-rDNA locus during growth, suggesting that their independence is a general feature (Supplementary Fig.\u0026nbsp;3). We showed that each single mutant caused a 4 to 5-fold increase in marker loss at rDNA, while the \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e double mutant led to a 10-fold increase (Supplementary Fig.\u0026nbsp;3a). Similarly, in the gross chromosome rearrangement (GCR) assay that assesses the stability of a non-rDNA locus, we detected a 59-fold increase of marker loss in the \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e double mutant and a 16 to 20-fold increase in the corresponding single mutants (Supplementary Fig.\u0026nbsp;3b). These genetic data are in line with the results from two cell cycle checkpoint assays described above as well as previous biochemical findings that Mms22 and Slx4 partner with Rtt107 in a mutually exclusive manner\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Together, they strongly suggest that Mms22 and Slx4 represent parallel pathways in regulating both the DNA damage checkpoint and genomic stability.\u003c/p\u003e \u003cp\u003eWhile our data support the functional independence of Mms22 and Slx4, the two proteins have the potential to indirectly affect each other due to sharing a common partner, Rtt107, that facilitates the chromatin recruitment of both\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. To examine this possibility, we asked if Mms22 and Slx4 affect each other\u0026rsquo;s chromatin association. We queried how the loss of the Slx4-Rtt107 interaction could affect Mms22 chromatin association and vice versa. We confirmed the reported findings that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e each reduced its own chromatin association (Fig.\u0026nbsp;3d). In contrast, \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e led to an increase in Slx4 level on chromatin while \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e showed no effect on the chromatin association of Mms22 (Fig.\u0026nbsp;3d and Supplementary Fig.\u0026nbsp;3c). These results suggest that the checkpoint dampening defects seen for \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells are not due to an indirect effect of reducing Slx4 chromatin association, and vice versa. This notion is consistent with data describing the additive effect of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e shown above (Figs.\u0026nbsp;3a-3c); together they suggest that Mms22 and Slx4 can work in parallel pathways with each collaborating with Rtt107 (Fig.\u0026nbsp;3e).\u003c/p\u003e\n\u003ch3\u003eThe Mms22-Rtt107 interaction and the Rtt101 E3 promote Rad9 degradation\u003c/h3\u003e\n\u003cp\u003eWe next investigated the mechanisms by which the Mms22-Rtt107 interaction can promote DDC dampening. As Mms22 is a subunit of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e ubiquitin E3\u003csup\u003e27\u003c/sup\u003e, a likely means for it to downregulate the checkpoint is through protein degradation. Given the strong genetic suppression of \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e by \u003cem\u003erad9\u003c/em\u003e and \u003cem\u003eddc1\u003c/em\u003e mutant alleles (Figs.\u0026nbsp;2a and 2c-2e, Supplementary Fig.\u0026nbsp;2), we examined the stability of these two proteins. We performed a standard procedure that utilizes cycloheximide (CHX) to block new protein synthesis, thus allowing the monitoring of protein stability during a time course. We first examined Ddc1 and Rad9 during normal growth. While the Ddc1 protein level showed minimal changes during an eight-hour time course, the Rad9 protein level exhibited a strong reduction over time (Figs.\u0026nbsp;4a and 4b, Supplementary Fig.\u0026nbsp;4a). Importantly, Rad9 instability is improved by the removal of Rtt101 or Rtt107, and by \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e (Fig.\u0026nbsp;4a). Similar improvement was also seen in \u003cem\u003emms22∆\u003c/em\u003e and \u003cem\u003emms1∆\u003c/em\u003e cells (Supplementary Fig.\u0026nbsp;4b). Rad9 protein level quantification based on at least two biological isolates per genotype showed that all examined mutants stabilized Rad9 levels to a similar degree (Fig.\u0026nbsp;4b and Supplementary Fig.\u0026nbsp;4c). In contrast to this group of mutants, Rad9 degradation was not affected by the lack of Slx4 (Supplementary Fig.\u0026nbsp;4d). We thus conclude that the Rtt107-Mms22-Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e axis but not the Rtt107-Slx4 axis affects Rad9 protein stability. As mutants of Rtt107, Mms22, and Rtt101 did not fully stabilize Rad9, additional means also exist to promote Rad9 degradation.\u003c/p\u003e \u003cp\u003eWe next monitored Rad9 stability when cells were treated by MMS. Again, we observed Rad9 degradation in wild-type cells and this was reduced upon the removal of Rtt107, Rtt101, and in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells (Figs.\u0026nbsp;4c and 4d). This group of mutants also showed persistent Rad9 phosphorylation, which is catalyzed by the Mec1 kinase and serves as a marker for DDC activation (Fig.\u0026nbsp;4c)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. These results are consistent with data presented above and further support the conclusion that the Mms22-Rtt107 interaction is required for degrading Rad9. Collectively, our findings suggest that the Mms22-Rtt107 interaction as well as the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 are partly responsible for Rad9 degradation.\u003c/p\u003e\n\u003ch3\u003eRad9 degradation is mediated by proteasomes\u003c/h3\u003e\n\u003cp\u003eTo further test the above notion, we asked whether Rad9 protein instability is mediated by the proteasomes. If Mms22 and Rtt107 collaborate with the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 in Rad9 degradation, we would expect that blocking proteasomal functions can hinder Rad9 degradation. We treated cells with MG132 that inhibits proteasomal activity, and with CHX that blocks new protein synthesis. In the control DMSO treatment, Rad9 protein level reduced during a four-hour time course (Fig.\u0026nbsp;5a). However, the Rad9 protein was stabilized in the presence of MG132 (Fig.\u0026nbsp;5a). This result suggests that Rad9 degradation is mediated by proteasomes. This conclusion is in line with the involvement of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 in Rad9 degradation in cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003erad9-K1088M\u003c/b\u003e \u003cb\u003erescues the MMS sensitivity caused by the loss of Rtt101 and Mms1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next examined the functional significance of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3\u0026rsquo;s involvement in Rad9 degradation. If this role is important for cell survival in genotoxins, we would expect that as seen for \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e mutant, genotoxin sensitivity caused by the loss of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 could be rescued by a mildly defective Rad9 allele. Indeed, we found that \u003cem\u003erad9-K1088M\u003c/em\u003e greatly increased the viability of either \u003cem\u003ertt101∆\u003c/em\u003e or \u003cem\u003emms1∆\u003c/em\u003e mutant cells on media containing MMS, CPT, or HU (Fig.\u0026nbsp;5b). This data provides evidence that like Mms22, Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3\u0026rsquo;s involvement in Rad9 degradation can also be important for cellular survival in the face of genotoxins.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMms22-Rtt107 interaction helps the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 associate with chromatin and regulating Rad9 stability on chromatin\u003c/h2\u003e \u003cp\u003eOur data thus far suggest that Rtt107 dampens DNA damage checkpoint signaling through binding to Mms22, in addition to binding to Slx4. Previous studies have shown that Rtt107 helps to recruit both Mms22 and Slx4 to chromatin via its ability to recognize γH2A\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. These findings raise the possibility that Rtt107 binding to Mms22 may help the chromatin association of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 for degrading Rad9 on chromatin.\u003c/p\u003e \u003cp\u003eWe tested the above notion first by examining the chromatin association of Rtt101 and Mms1 when the Mms22-Rtt107 interaction is disrupted by \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e. We used a well-established chromatin fraction method to separate chromatin and soluble fractions\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. We found that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e not only reduced its own chromatin association but also lessened those for Rtt101 and Mms1 (Fig.\u0026nbsp;6a). We moved on to monitor Rad9 levels on chromatin and found that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells exhibited an increased amount of Rad9 on chromatin (Fig.\u0026nbsp;6b), suggesting that the Mms22-Rtt107 interaction is required for Rad9 loss from chromatin. Importantly, in the presence of CHX that allows the examination of protein degradation, we found that degradation of Rad9 in the chromatin fraction was largely blocked in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells (Fig.\u0026nbsp;6c). The Rad9 stabilization effect conferred by \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e is much stronger for the chromatin pools of Rad9 compared with the whole cell extract (WCE) pool of Rad9 (Fig.\u0026nbsp;6c), suggesting that the main effect of Mms22-Rtt107 stems from the regulation of the stability of Rad9 in the chromatin fraction. Collectively, these data provide evidence that the Mms22-Rtt107 interaction is important for the chromatin association of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 and degrading Rad9 in the chromatin fraction.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMms22-Rtt107 binding does not affect the Rad53-Asf1 or Rtt107-Dpb11 association\u003c/h3\u003e\n\u003cp\u003eThe Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 was previously shown to collaborate with Asf1 in down-regulating DDC when cells suffer from two double-strand DNA breaks (DSBs)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The proposed model suggests that Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 may help Asf1 binding to Rad53, an interaction that could disfavor Rad53 phosphorylation thus leading to reduced DDC levels\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To discern if the Mms22 collaboration with Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e during DDC dampening might be related to Asf1-Rad53 association, we examined this interaction by co-immunoprecipitation. We found that the amount of Asf1 recovered from Rad53 co-immunoprecipitation was similar between \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e and wild-type cells, in both normal growth and MMS treated conditions (Supplementary Fig.\u0026nbsp;5a). This result suggests that the effect of the Mms22-Rtt107 interaction on DDC dampening is not via regulating the Asf1-Rad53 interaction. Finally, considering that Rtt107 also associates with another checkpoint factor, Dpb11\u003csup\u003e23\u003c/sup\u003e, we asked whether this interaction is perturbed in \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells. We found that while \u003cem\u003emms22-RIM\u003c/em\u003e disrupted the Rtt107-Mms22 interaction, it did not affect the Rtt107-Dpb11 association in the presence or absence of MMS treatment (Supplementary Fig.\u0026nbsp;5b). These results suggest the DDC recovery role of the Rtt107-Mms22 interaction is not mediated by altering the Rtt107-Dpb11 interaction.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCellular survival of genotoxin treatments depends on the induction of the DNA damage checkpoint as well as its subsequent downregulation. While many studies have examined DDC induction, DDC downregulation is less understood. Here we describe a DDC dampening strategy that is mediated by the degradation of a central DDC mediator protein, Rad9, in yeast. We identified the players that contribute to this regulation, including the scaffold protein Rtt107, the Mms22 substrate adapter of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e ubiquitin E3, and the ubiquitin E3 itself. Results from genetic suppression and checkpoint assays strongly support the conclusion that the Mms22-Rtt107 interaction provides an important means to downregulate DDC. Further, biochemical evidence suggests that Mms22 and Rtt107 collaborate with the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 to facilitate proteasome-mediated degradation of Rad9. Interestingly, this regulation was more prominently seen for the chromatin pool of Rad9 compared with the non-chromatin pool. The preferential effect can be explained by our finding that the Mms22-Rtt107 interaction promotes the chromatin association of the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3. Based on these data, we propose a model wherein Rtt107 recruits the Mms22-containing Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 to chromatin to facilitate Rad9 degradation (Fig.\u0026nbsp;6d). Chromatin targeting of the Mms22-containing E3 could be achieved through previously identified bifurcated interactions of Rtt107, with its tetra-BRCT domain binding to Mms22 and additional binding sites recognizing γH2A and the phosphorylated H4T80 mark\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Finally, we provide evidence to support the notion that the Rtt107-Mms22-Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 axis acts in parallel with the Rtt107-Slx4 axis that competes with Rad9 for binding to nucleosomes. As such, Rtt107 could coordinate a two-pronged strategy that exploits distinct anti-Rad9 effects to downregulate DDC (Fig.\u0026nbsp;6d).\u003c/p\u003e \u003cp\u003eRtt107 is required for chromatin targeting of both Mms22 and Slx4, which compete for the same binding site on the Rtt107 tetra-BRCT domain\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Despite the competition, both the Mms22-Rtt107 and Slx4-Rtt107 complexes are detected in cells\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Thus, Rtt107 is likely present in sufficient amounts to allow the formation of the two complexes. In line with this notion, the loss of Slx4-Rtt107 binding had no effect on the abundance of Mms22 on chromatin (Fig.\u0026nbsp;3d and Supplementary Fig.\u0026nbsp;3c). However, disrupting the Mms22-Rtt107 association increased Slx4 levels on chromatin (Fig.\u0026nbsp;3d and Supplementary Fig.\u0026nbsp;3c). These observations suggest a unidirectional modulation of the dynamics of the Rtt107 interactome that is worthwhile for further testing in the future. Most relevant here, despite showing increased chromatin association of Slx4, which could favor DDC downregulation, \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e cells nevertheless showed persistent checkpoint (Fig.\u0026nbsp;3d and Supplementary Fig.\u0026nbsp;3c). These data support our conclusion that the DDC dampening defect caused by \u003cem\u003emms22\u003c/em\u003e mutants is not due to lessening Slx4 chromatin association and vice versa. Rather, Mms22 and Slx4 each employ a different strategy to downregulate the checkpoint. While the Slx4-Rtt107 axis can disfavor Rad9 binding to chromatin\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, our result suggests that the Mms22-Rtt107 axis regulates Rad9 degradation. Interestingly, while \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e led to a general increase in Rad9 stability, it had a more prominent effect on the chromatin pool of Rad9, which is required for DDC signaling (Fig.\u0026nbsp;6c). This makes it possible that targeting this pool of Rad9 can make the Mms22-Rtt107 axis more efficient in downregulating the checkpoint. We note that we cannot exclude additional roles of Mms22-Rtt107 in affecting Rad9 association with chromatin. Nonetheless, since Slx4 did not affect Rad9 stability, regulation of Rad9 stability appears to be rather specific to the Mms22-Rtt107 axis and does not reflect a general feature of DDC dampening factors.\u003c/p\u003e \u003cp\u003eUsing a Slx4 mutant specifically abolishing its binding to Rtt107, our data strengthened the previous conclusions regarding the role of Slx4 and Rtt107 in checkpoint dampening. Further, we show that the Slx4-Rtt107 interaction had a stronger effect when the Mms22-Rtt107 interaction was lost (Figs.\u0026nbsp;3a and 3b). In addition, \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e cells showed slower S phase progression compared with wild-type cells. This effect can be related to findings that Slx4 and Rtt107 associate with chromatin behind the replication fork to help replication in MMS conditions\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e had no obvious defect in S phase progress but delayed exiting from the G2/M phase and increased levels of Rad53 activation (Figs.\u0026nbsp;3a and 3b). These observations provide additional support for the conclusion that Rtt107 can participate in two DDC regulatory pathways involving either Mms22 that mainly affects G2/M exit or involving Slx4 that has roles in controlling S phase progression.\u003c/p\u003e \u003cp\u003eThe proteins examined here, including Rtt107, Mms22, Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and Slx4 are all multi-functional and each has several binding partners\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In addition to our findings here, the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 has been suggested to downregulate DDC via regulating the Asf1-Rad53 interaction\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Using the \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e separation-of-function allele, we showed that the Rtt107-Mms22 collaboration with Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 does not affect the Asf1-Rad53 association (Supplementary Fig.\u0026nbsp;5a). These data provide evidence that the Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 has at least two roles in checkpoint dampening, one through Rad9 degradation and the other via regulating the Asf1-Rad53 interaction. We also found that \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e did not affect the Rtt107 interaction with another checkpoint mediator Dbp11 known to also bind to Slx4 (Supplementary Fig.\u0026nbsp;5b). These data highlight the importance of using separation-of-function alleles in clarifying the role(s) of complex protein-protein interactions employed in DDC and genome maintenance.\u003c/p\u003e \u003cp\u003eOur data suggest that additional ubiquitin E3(s) can also help proteasome-mediated degradation of Rad9. It is common that one substrate is subjected to multiple E3 regulations to ensure their optimal degradation. Future studies aiming to identify additional factors involved in Rad9 degradation can expand our understanding of protein-level control of this key checkpoint mediator. Though our work focuses on the role of the Mms22-Rtt107 interaction in DNA damage conditions, we found that disruption of this interaction by \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e also affects genome maintenance during growth. In particular, \u003cem\u003emms22\u003c/em\u003e\u003csup\u003e\u003cem\u003eRIM\u003c/em\u003e\u003c/sup\u003e led to increased genomic instability at both rDNA and non-rDNA sites in the absence of genotoxin and showed additive effect with \u003cem\u003eslx4\u003c/em\u003e\u003csup\u003e\u003cem\u003eTTS\u003c/em\u003e\u003c/sup\u003e (Supplementary Figs.\u0026nbsp;3a and 3b). This genetic finding corroborates the biochemical data that Rad9 stability is similarly regulated by Mms22 during growth and under genotoxin treatment, suggesting that Rad9 is a general client for the Mms22-Rtt101\u003csup\u003eMms\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e E3 and its degradation is not triggered by DDC (Figs.\u0026nbsp;4a and 4b). Together, these data raise the possibility that the genome-protecting roles of the Rtt107-Mms22 axis during growth are also related to limiting Rad9 levels. It is currently unclear how limiting Rad9 levels during growth is beneficial to cells. Given that the Mec1 kinase shows local activation but does not trigger global DDC during normal S phase, we speculate that Rad9 degradation may help restrain Mec1 activation to a local level, thus preventing unnecessary and harmful full-on DDC\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In this scenario, DDC dampening mechanisms may not only be critical for long-term survival in genomic stress conditions but could also help manage genome stability during growth. Future studies to explore these possibilities can provide a fuller picture to better unite DDC regulation in both conditions. Given the conservation of the protein factors examined here, results of our study can stimulate the studies of how higher eukaryotes can utilize protein degradation as a tool to downregulate checkpoint, and of the functional relevance of such regulation in tumorigenesis and during normal development.\u003c/p\u003e "},{"header":"METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003cp\u003e \u003cb\u003eYeast strains and genetic manipulation.\u003c/b\u003e Yeast strains used in this work are listed in Table S1 and they are derivatives of W1588-4C, a \u003cem\u003eRAD5\u003c/em\u003e variety of W303 (\u003cem\u003eMATa ade2-1 can1-100 ura3-1 his3-11,15 leu2-3,112 trp1-1 rad5-535\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Protein tagging, gene deletion, and mutant alleles were generated at the endogenous genomic loci following standard PCR-based or CRISPR\u0026ndash;Cas9 based methods\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. All genetically altered loci were verified by sequencing. Standard yeast genetic manipulation was used for tetrad analyses and all experiments were conducted at 30\u0026ordm;C unless noted. At least two different biological isolates per genotype were used for each assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenotoxin sensitivity tests.\u003c/b\u003e Spotting assays to detect DNA damage sensitivity were carried out as described previously\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In brief, yeast cultures were grown overnight at 30\u0026deg;C in rich medium (YPD). Cultures were then diluted to OD\u003csub\u003e600\u003c/sub\u003e 0.15 and allowed to grow to early mid-log phase in YPD. Subsequently, cells were spotted in 10-fold serial dilutions on plates containing YPD with or without DNA damaging agents at the indicated concentrations. Plates were incubated at 30\u0026deg;C and photographed after 2\u0026ndash;4 days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynchronization and cell cycle analysis.\u003c/b\u003e The cell synchronization procedure was used as previously described with a few modifications\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In brief, early-mid log-phase yeast cultures grown in the YPD media at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.2 were treated with α factor for G1 arrest. After the initial addition of 5 \u0026micro;g/ml α factor (Thermo Fisher) for one hour, a second dose of 2.5 \u0026micro;g/ml α factor was added for another hour. Cell morphology was monitored to confirm G1 arrest. Subsequently, cells were released from G1 arrest by growing in YPD media containing 100 \u0026micro;g/ml protease (Sigma) to degrade α factor. The media also contained 0.03% MMS for genotoxin treatment. After 45 min, MMS was washed out, and cells were allowed to grow in YPD media. Samples were taken every 20 min for FACS analyses and Rad53 examination. FACS analyses were done as previously described\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Briefly, cells were fixed in 70% ethanol for 1 h at room temperature or overnight at 4\u0026deg;C. RNAs and proteins were degraded by sequential treatment with RNase A (Sigma) and Protease K (Sigma). DNA was stained with Sytox Green (Invitrogen). Samples were examined using a BD LSR II flow cytometer (BD). Data analyses were performed using the FlowJo Software (BD).\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein extraction to examine Rad53 phosphorylation.\u003c/b\u003e Protein extracts were made using a TCA (Trichloroacetic acid) method to maintain Rad53 phosphorylation as described previously\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In brief, cells were homogenized using 0.5 mm silica beads (BioSpec Products) in the presence of 20% TCA on a FastPrep-24 bead-beating grinder (MP Biomedicals). After removing the supernatant by centrifugation, the precipitated proteins were washed with 5% cold TCA, and then dissolved in protein loading buffer (50 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.05% bromophenol blue). Samples were boiled for 5 min before being loaded onto 4\u0026ndash;20% SDS-PAGE gels (Bio-Rad) and subsequently examined by immunoblotting (see below).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-immunoprecipitation.\u003c/b\u003e Standard protocols were followed to perform co-immunoprecipitation. In brief, cells were disrupted by bead beating in lysis buffer that contained 20 mM HEPES-KOH (pH 7.5), 100 mM KOAc, 1% Triton X-100, 2 mM Mg(OAc)\u003csub\u003e2\u003c/sub\u003e, 1 mM NaF, 2 mM β-glycerophosphate, and EDTA-free protease inhibitors (Roche). Lysates were cleaned by centrifugation at 20,000g for 30 min to obtain whole-cell extract, which was then incubated with anti-Flag beads (Sigma-Aldrich) or anti-HA beads (Pierce) for 2 h at 4\u0026deg;C. After washing the beads three times with lysis buffer, bead-bound proteins were eluted using loading buffer as described above. Proteins were boiled for 5 min before being loaded onto 4\u0026ndash;20% gradient gels (Bio-Rad) for electrophoresis and immunoblotting (see below).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoblotting and antibodies used.\u003c/b\u003e Proteins were transferred from gels to 0.2 \u0026micro;m nitrocellulose membranes (GE) before immunoblotting. The antibodies used include: anti-phosphorylated active Rad53 (F9, a gift from Marco Foiani and Daniele Piccini), anti-Ddc1 (a gift from Marco Muzi-Falconi), anti-Dpb11 (a gift from Dirk Remus), anti-Rad9 (a gift from John Petrini), anti-Histone H3 (ab1791, Abcam), anti-Actin (C4, 8691001, MP Biomedicals), anti-Sir2 (yN-19, sc-6666, Santa Cruz), anti-Flag (M2, F1804, Sigma), anti-HA (F-7, sc-7392, Santa Cruz), anti-Myc (9E10, BE0238, Bio X Cell), anti-TAP (PAP, P1291, Sigma), anti-Pgk1 (22C5, Molecular Probes), and anti-Tubulin (YL1/2, ab6160, Abcam),\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromatin fractionation.\u003c/b\u003e Chromatin fractionation was performed as described previously with a few modifications\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In brief, yeast spheroplasts derived from log-phase cells were lysed using extraction buffer (150 mM KOAc, 20 mM pH 6.6 PIPES-KOH, 2 mM Mg(OAc)\u003csub\u003e2\u003c/sub\u003e, 1 mM NaF, 0.5 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) by incubating on ice for 5 min in the presence Zymolyase. Lysates were then subjected to centrifugation at 16,000 g for 15 min at 4\u0026deg;C on a sucrose cushion. The resultant chromatin pellets were washed and resuspended with extraction buffer. Loading buffer was added to each sample before boiling for 5 min. Samples were then examined by SDS-PAGE followed by immunoblotting. Tubulin or Pgk1 was used as a marker of the non-chromatin fraction, while histone H3 or Sir2 was used as a marker for chromatin-associated proteins.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein stability assay.\u003c/b\u003e Protein stability was examined using a standard protocol as described previously\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Briefly, yeast cultures were grown to OD\u003csub\u003e600\u003c/sub\u003e 0.2 before CHX was added to a final concentration of 100 \u0026micro;g/ml to inhibit protein synthesis. Equal numbers of cells were collected every 1 h following CHX addition. Cell lysates were examined using the TCA method described above. The band intensity of each protein after immunoblotting was quantified using the FIJI software\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003erDNA marker loss and GCR assays\u003c/b\u003e. Standard protocols were followed for both assays. For the GCR assay that assesses the loss of the \u003cem\u003eURA3\u003c/em\u003e and \u003cem\u003eCAN1\u003c/em\u003e markers inserted at the YEL068c locus, at least six cultures were examined for each genotype\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Cells were plated on SC (synthetic complete) media containing 5-FOA to counter-select \u003cem\u003eURA3\u003c/em\u003e and containing canavanine to counter-select \u003cem\u003eCAN1\u003c/em\u003e. The numbers of colonies grown on 5-FOA- containing SC (FC) plates reflect those that lost \u003cem\u003eURA3\u003c/em\u003e and \u003cem\u003eCAN1\u003c/em\u003e markers. Cells were also plated on SC plates to determine the total viable colonies. GCR rates were calculated as \u003cem\u003em/N\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e by the following formula: m[1.24\u0026thinsp;+\u0026thinsp;ln(m)]\u0026thinsp;=\u0026thinsp;N\u003csub\u003eFC\u003c/sub\u003e. Here, m represents mutational events, N\u003csub\u003eFC\u003c/sub\u003e is the number of colonies on FC plates, and N\u003csub\u003eT\u003c/sub\u003e is the number of colonies on SC plates. The upper and lower 95% confidence intervals were derived. Frequencies for the loss of the \u003cem\u003eADE2\u003c/em\u003e and \u003cem\u003eCAN1\u003c/em\u003e markers inserted into the rDNA array were measured as described previously\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Cells were grown to stationary phase over equal doubling times and plated on SC media to count total viable colonies. Cells were also plated on media containing canavanine (SC\u0026thinsp;+\u0026thinsp;Can) to select those that lost the \u003cem\u003eCAN1\u003c/em\u003e marker. The frequency of marker loss was calculated as: F\u0026thinsp;=\u0026thinsp;N\u003csub\u003ecan\u003c/sub\u003e/N\u003csub\u003eC\u003c/sub\u003e, where N\u003csub\u003eCan\u003c/sub\u003e is the number of colonies on SC\u0026thinsp;+\u0026thinsp;Can plates and N\u003csub\u003eC\u003c/sub\u003e is the number of cells plated on SC plates\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTION:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors were involved in research design and data analyses. B.W., D.G., S.L, and T.C.E performed experiments. B.W. and X.Z. wrote the paper with all the authors\u0026rsquo; input.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Marco Foiani, Daniele Piccini, Marco Muzi-Falconi, Dirk Remus and John Petrini for sharing antibodies. B. Wan. is supported by NSFC grant 32170087. X.Z. is supported by National Institute of General Medical Sciences (NIGMS) grants R35GM145260. T.C.E is supported by a Kravis WiSE Fellowship. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHartwell LH, Weinert TA (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246:629\u0026ndash;634\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnoch T, Carr AM, Nurse P (1992) Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev 6:2035\u0026ndash;2046\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanz MC, Dibitetto D, Smolka MB (2019) DNA damage kinase signaling: checkpoint and repair at 30 years. EMBO J 38:e101801\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUsui T, Ogawa H, Petrini JH (2001) A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol Cell 7:1255\u0026ndash;1266\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300:1542\u0026ndash;1548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElledge SJ (1996) Cell cycle checkpoints: preventing an identity crisis. Science 274:1664\u0026ndash;1672\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoiani M et al (2000) DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae. Mutat Res 451:187\u0026ndash;196\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrison JC, Haber JE (2006) Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40:209\u0026ndash;235\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaca VM et al (2020) Maximized quantitative phosphoproteomics allows high confidence dissection of the DNA damage signaling network. Sci Rep 10:18056\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNyberg KA, Michelson RJ, Putnam CW, Weinert TA (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 36:617\u0026ndash;656\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClemenson C, Marsolier-Kergoat MC (2009) DNA damage checkpoint inactivation: adaptation and recovery. DNA Repair (Amst) 8:1101\u0026ndash;1109\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaterman DP, Haber JE, Smolka MB (2020) Checkpoint responses to DNA double-strand breaks. Annu Rev Biochem 89:103\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePizzul P et al (2022) The DNA damage checkpoint: A tale from budding yeast. Front Genet 13:995163\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhouo PY, Bastos de Oliveira FM, Liu Y, Ma CJ, Smolka MB (2013) DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9. Nature 493:120\u0026ndash;124\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGobbini E et al (2015) Sae2 function at DNA double-strand breaks Is bypassed by dampening Tel1 or Rad53 activity. PLoS Genet 11:e1005685\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu TY, Kimble MT, Symington LS (2018) Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 115, E11961-E11969\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhingra N et al (2021) The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 118\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePizzul P et al (2024) Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding. Nucleic Acids Res 52:2355\u0026ndash;2371\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWysocki R et al (2005) Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9. Mol Cell Biol 25:8430\u0026ndash;8443\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHammet A, Magill C, Heierhorst J, Jackson SP (2007) Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast. EMBO Rep 8:851\u0026ndash;857\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVialard JE, Gilbert CS, Green CM, Lowndes NF (1998) The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J 17:5679\u0026ndash;5688\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez Y et al (1996) Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271:357\u0026ndash;360\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhouo PY, Bastos de Oliveira FM, Almeida BS, Smolka MB (2010) DNA damage signaling recruits the Rtt107-Slx4 scaffolds via Dpb11 to mediate replication stress response. Mol Cell 39:300\u0026ndash;306\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChin JK, Bashkirov VI, Heyer WD, Romesberg FE (2006) Esc4/Rtt107 and the control of recombination during replication. DNA Repair (Amst) 5:618\u0026ndash;628\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMimura S et al (2010) Cul8/Rtt101 forms a variety of protein complexes that regulate DNA damage response and transcriptional silencing. J Biol Chem 285:9858\u0026ndash;9867\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHang LE et al (2015) Rtt107 is a multi-functional scaffold supporting replication progression with partner SUMO and ubiquitin ligases. Mol Cell 60:268\u0026ndash;279\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaidi IW et al (2008) Rtt101 and Mms1 in budding yeast form a CUL4(DDB1)-like ubiquitin ligase that promotes replication through damaged DNA. EMBO Rep 9:1034\u0026ndash;1040\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan B, Hang LE, Zhao X (2016) Multi-BRCT scaffolds use distinct strategies to support genome maintenance. Cell cycle (Georgetown Tex) 15:2561\u0026ndash;2570\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan B, Wu J, Meng X, Lei M, Zhao X (2019) Molecular basis for control of diverse genome stability factors by the multi-BRCT scaffold Rtt107. \u003cem\u003eMol. Cell\u003c/em\u003e 75, 238\u0026ndash;251 e235\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfander B, Matos J (2017) Control of Mus81 nuclease during the cell cycle. FEBS Lett 591:2048\u0026ndash;2056\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBermejo R et al (2007) Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev 21:1921\u0026ndash;1936\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuddu F et al (2008) Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint. Mol Cell Biol 28:4782\u0026ndash;4793\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsborn AJ, Elledge SJ (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev 17:1755\u0026ndash;1767\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaciotti V, Clerici M, Scotti M, Lucchini G, Longhese MP (2001) Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway. Mol Cell Biol 21:3913\u0026ndash;3925\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmili A (1998) MEC1-dependent phosphorylation of Rad9p in response to DNA damage. Mol Cell 2:183\u0026ndash;189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X et al (2012) Structure of C-terminal tandem BRCT repeats of Rtt107 protein reveals critical role in interaction with phosphorylated histone H2A during DNA damage repair. J Biol Chem 287:9137\u0026ndash;9146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalint A et al (2015) Assembly of Slx4 signaling complexes behind DNA replication forks. EMBO J 34:2182\u0026ndash;2197\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonner JN et al (2016) Smc5/6 mediated sumoylation of the Sgs1-Top3-Rmi1 complex promotes removal of recombination intermediates. Cell Rep 16:368\u0026ndash;378\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsabar M et al (2016) Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair. Genes Dev 30:1211\u0026ndash;1224\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMillan-Zambrano G et al (2018) Phosphorylation of histone H4T80 triggers DNA damage checkpoint recovery. Mol Cell 72:625\u0026ndash;635e624\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCussiol JR, Dibitetto D, Pellicioli A, Smolka MB (2017) Slx4 scaffolding in homologous recombination and checkpoint control: lessons from yeast. Chromosoma 126:45\u0026ndash;58\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanz MC et al (2018) Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling. Genes Dev 32:822\u0026ndash;835\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Blobel G (2005) A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 102, 4777\u0026ndash;4782\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng X et al (2020) DNA polymerase epsilon relies on a unique domain for efficient replisome assembly and strand synthesis. Nat Commun 11:2437\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei L, Zhao X (2016) A new MCM modification cycle regulates DNA replication initiation. Nat Struct Mol Biol 23:209\u0026ndash;216\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S et al (2021) Esc2 orchestrates substrate-specific sumoylation by acting as a SUMO E2 cofactor in genome maintenance. Genes Dev 35:261\u0026ndash;272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung I, Zhao X (2015) DNA break-induced sumoylation is enabled by collaboration between a SUMO ligase and the ssDNA-binding complex RPA. Genes Dev 29:1593\u0026ndash;1598\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelle A, Tanay A, Bitincka L, Shamir R, O'Shea EK (2006) Quantification of protein half-lives in the budding yeast proteome. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e 103, 13004\u0026ndash;13009\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan J et al (2010) Ubiquitylation of FACT by the cullin-E3 ligase Rtt101 connects FACT to DNA replication. Genes Dev 24:1485\u0026ndash;1490\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676\u0026ndash;682\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePutnam CD, Kolodner RD (2010) Determination of gross chromosomal rearrangement rates. \u003cem\u003eCold Spring Harb Protoc\u003c/em\u003e pdb prot5492 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFritze CE, Verschueren K, Strich R (1997) Easton Esposito, R. Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA. EMBO J 16:6495\u0026ndash;6509\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRegan-Mochrie G et al (2022) Yeast ORC sumoylation status fine-tunes origin licensing. Genes Dev 36:807\u0026ndash;821\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Muller EG, Rothstein R (1998) A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell 2:329\u0026ndash;340\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DNA damage checkpoint, checkpoint dampening, Rtt107, Mms22, Slx4","lastPublishedDoi":"10.21203/rs.3.rs-4417144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4417144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe DNA damage checkpoint is a highly conserved signaling pathway induced by genotoxin exposure or endogenous genome stress. It alters many cellular processes such as arresting the cell cycle progression and increasing DNA repair capacities. However, cells can downregulate the checkpoint after prolonged stress exposure to allow continued growth and alternative repair. Strategies that can dampen the DNA damage checkpoint are not well understood. Here, we report that budding yeast employs a pathway composed of the scaffold protein Rtt107, its binding partner Mms22, and an Mms22-associated ubiquitin ligase complex to downregulate the DNA damage checkpoint. Mechanistically, this pathway promotes the proteasomal degradation of a key checkpoint factor, Rad9. Furthermore, Rtt107 binding to Mms22 helps to enrich the ubiquitin ligase complex on chromatin and target the chromatin-bound form of Rad9. Finally, we provide evidence that the Rtt107-Mms22 axis operates in parallel with the Rtt107-Slx4 axis, which displaces Rad9 from chromatin. We thus propose that Rtt107 enables a bifurcated \u0026ldquo;anti-Rad9\u0026rdquo; strategy to optimally downregulate the DNA damage checkpoint.\u003c/p\u003e","manuscriptTitle":"The Mms22-Rtt107 axis dampens the DNA damage checkpoint by reducing the stability of the Rad9 checkpoint mediator","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-24 06:57:07","doi":"10.21203/rs.3.rs-4417144/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"adebcc19-f837-4d69-9a5e-ebfb6c854929","owner":[],"postedDate":"May 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32330853,"name":"Biological sciences/Molecular biology/DNA damage and repair/DNA damage checkpoints"},{"id":32330854,"name":"Biological sciences/Molecular biology/DNA damage and repair/DNA damage response"},{"id":32330855,"name":"Biological sciences/Genetics/Genomic instability"}],"tags":[],"updatedAt":"2025-01-04T08:15:59+00:00","versionOfRecord":{"articleIdentity":"rs-4417144","link":"https://doi.org/10.1038/s41467-024-54624-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-02 05:00:00","publishedOnDateReadable":"January 2nd, 2025"},"versionCreatedAt":"2024-05-24 06:57:07","video":"","vorDoi":"10.1038/s41467-024-54624-0","vorDoiUrl":"https://doi.org/10.1038/s41467-024-54624-0","workflowStages":[]},"version":"v1","identity":"rs-4417144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4417144","identity":"rs-4417144","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}