EXO1 is associated with Ku-independent DNA double strand break repair pathway and the inhibition of EXO1 chemosensitizes DDP resistance lung cancer cells

EXO1 is associated with Ku-independent DNA double strand break repair pathway and the inhibition of EXO1 chemosensitizes DDP resistance lung cancer cells | 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 Research Article EXO1 is associated with Ku-independent DNA double strand break repair pathway and the inhibition of EXO1 chemosensitizes DDP resistance lung cancer cells Yonghong Wang, Yalin Zhu, Hui Zhang, Zhenglan Huang, Wenli Feng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8486386/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alternative end joining (Alt-EJ) is an error-prone repair pathway of DNA double-strand break (DSB) which functions independent of nonhomologous end joining (NHEJ) core factors, especially Ku protein. As a less studied repair pathway, but not less important one, its involving proteins need further investigation. By using single-stranded DNA oligomers (ssO) displacing Ku protein from damaged DNA ends and facilitating Ku independent DSB repair proteins recruited to DNA ends, we identify EXO1 exonuclease is associated with Alt-EJ and may consider as a novel player participating in Alt-EJ pathway in Ku70-deficient lung cancer cells. Depletion of EXO1 greatly increased cellular sensitivity to Cisplatin (DDP) and DNA-dependent protein kinase (DNA-PK) inhibitor NU7026 in vitro due to the inhibition of the repair of DSBs. EXO1 inhibition also attenuated the growth of DPP-resistant tumors in the in vivo mouse model through increasing expression of apoptotic protein caspase-3 and 53BP1. Our results revealed EXO1 participation in DSB repair by Alt-EJ. Suppression of EXO1 provides a chemosensitizing therapeutic option for lung cancer patients of DDP resistance. Alt-EJ EXO1 Cisplatin DNA double-strand break lung cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Double-strand breaks (DSBs) are the most lethal type of DNA damage, which are induced by endogenous cellular processes, such as V(D)J recombination and oxidative metabolism, as well as exogenous agents like ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors. In mammalian cells, there are two major repair pathways for DSBs, homologous recombination (HR) and canonical non-homologous end joining (C-NHEJ). The former requires sister chromatin as the template to complete high-fidelity repair. The C-NHEJ operates through the whole cell cycle, and is initiated by the binding of Ku70/Ku80 heterodimer, followed by DNA-dependent protein kinase catalytic subunits (DNA-PKcs), DNA ligase 4 (Lig4), XRCC4, XRCC4-like factor (XLF) etc. Ku provides protection of DNA ends from degradation and recruits other repair proteins, which make Ku the most important protein in maintenance of C-NHEJ pathway. While C-NHEJ is unavailable, especially in the absence of Ku from DNA ends, an error-prone alternative end joining (Alt-EJ) takes over as a backup option. It functions independent of nonhomologous end joining (NHEJ) or HR core components and is highly relevant to the chromosomal instability associated with cancer. In the past decades, proteins like poly (ADP-ribose) polymerase 1 (PARP-1), DNA Ligase 1 (Lig1), XRCC1/DNA Lig-3, C-terminal binding interaction protein (CtIP), DNA polymerase theta (POLQ) etc. have been identified participating in the pathway. More unrevealed proteins are on the way of being discovered. Whether Alt-EJ is an independent pathway remains controversial. Unlike other classical repair pathway, it forms a bricolage at DNA ends 1 by sharing players also involved in some other repair pathways, like PARP-1 in base-excision repair pathway for DNA single-strand breaks (SSBs) repair and MRN complex and CtIP of initial resection step of HR. Another feature of the Alt-EJ pathway is the formation of a high frequency of microhomologies and information deletions at ligated ends 2 , which increases the likelihood of gene mutations and leads to chromosomal instability. Notably, apart from tumorigenesis, some evidence revealed that it might aide to generate and reinforce the resistance to anticancer drugs. For example, in chronic myeloid leukemia (CML), it’s a probable cause for BCR-ABL gene mutations that help to form the resistance to BCR-ABL inhibitors 3 . Moreover, the increased contribution of the error-prone Alt-EJ pathway to anticancer drugs resistance is at least partially due to increased expression levels of the known error-prone Alt-EJ factors. For instance, DNA Lig3 and PARP-1 display higher steady state levels in tyrosine kinase inhibitor-resistant CML 4 and therapy-resistant breast cancer cell lines 5 . Importantly, survival of HR deficient tumors is promoted by mutagenic Alt-EJ DNA repair pathway 6 , 7 . Knockdown of POLQ is highly effective in killing HR-deficient cells, raising the possibility that cell proliferation in the absence of HR is dependent on Alt-EJ 7 . In anticancer chemotherapy, one of major mechanisms of Cisplatin (DDP) resistance is DNA damage repair, due the highly efficient repair system and elevated DNA repair capacity after cell damage, which enables cancer cells exhibit intrinsic resistance and undermines the efficacy of drug cytotoxicity. The known relevant repair pathways include nucleotide excision repair (NER), interstrand cross-links (ICLs), mismatch repair(MMR)and HR. In some cell lines, like ovarian A2780 cells, the DNA repair mechanism is not the major cause of DDP resistance. In cell lines that are sensitive to DDP, the DNA repair ability is weaker than that in cells resistant to DDP 8 . DNA double-strand break repair by HR begins with nucleolytic resection of the 5' DNA strand at the break ends. The end resection comprises the generation of relative short stretches of ssDNA by MRN and CtIP and extensive resection by EXO1 and BLM-DNA2. The latter has been considered to favor HR 9 . However, Alt-EJ shares initial resection step with HR which uses DNA resection to reveal single-stranded MHs for annealing before joining. Resection proteins like MRN complex and CtIP are versatile and have been demonstrated participating in C-NHEJ, HR and Alt-EJ 10–15 . Overexpression of EXO1 induces hyper-resection to attenuate both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability 16 . The regulation of EXO1 is critical for accurate end joining and the mechanism of resection is far from fully understood, whether EXO1 participate in Alt-EJ worth an investigation. Given Ku is considered the most essential factor to keep Alt-EJ marginal and remain the a faithful repair, in our previous work, we established a novel Alt-EJ model in which Ku can be hijacked by short single-stranded oligonucleotides (ssO) generated from artificial interference or DNA end resection 17 . The preventing of Ku accumulation DSBs ends favors Alt-EJ and make instant activation of Alt-EJ possible. This discover shed a light into the possibility that Alt-EJ can be locally activated at some DNA ends where Ku is relatively lacks and eventually lead to the chromosomal instability and locally initiation of cancer. Based on this model, in this study, Alt-EJ was favored at ends of biotinylated probe dsDNA in the presence of ssO in cell extracts in vitro . Damage repair related proteins participated in Alt-EJ was harvested in pull-down assay, screened with mass spectrometry analysis, and selected with RT-qPCR and western blotting. The chromatin binding affinity of candidate proteins was examined by biochemical fractionation and by immunostaining. EXO1, as one of our selected Alt-EJ protein, is found recruited to the DSB sites upon DNA damage in a C-NHEJ independent manner in Ku70-deficient lung cancer cells, in which Ku70 was down-regulated by using a ADV1-Ku70-192 virus to active Alt-EJ. Using a reporter plasmid assay in cells 18 – 20 , Alt-EJ was found decreased and C-NHEJ was increased in EXO1-depleted cells. Evaluated in SCGE analysis, EXO1-depleted cells exhibited higher sensitivity to DDP or DNA-PK inhibitor by formation of more DSBs. In xenograft model, combination therapy with DDP and EXO1-i significantly increased tumor inhibition rates. In our condition, we found that, in Ku70-deficient cancer cells, the chromatin-binding affinity of EXO1 increased in response to DSBs in C-NHEJ independent manner, revealing its role in Alt-EJ-mediated DNA repair. By regulating the repair pathway, it promotes cell survival, mediates cell resistance to chemotherapy agent DDP and promotes DSBs formation at DNA end. Herein, EXO1 is a potential candidate for developing lung cancer-specific drugs and customized targeted molecular therapy. 2. Materials and Methods 2.1. Reagents Calicheamicin-γ1 (Cali), purchased from MedChemExpress (Monmouth junction, NJ, USA), was dissolved in DMSO at 4 mM and stored at -80°C. NU7026 was also purchased from MedChemExpress (Monmouth junction, NJ, USA) and dissolved in DMSO. 2.2. Cell culture HBE, H520, A549, and A549/DDP drug resistence cell lines were purchased from the China Center for Type Culture Collection (Wuhan, China) and were grown in Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA) containing penicillin-streptomycin and 10% fetal bovine serum (FBS, Gibco, USA). 2.3. DNA fragments and oligonucleotides The sequence of the 75-bp DNA fragment used in pull-down experiments is as follows: 5′-CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC-3′, 5′-GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACG-3′. The sequence of the single-stranded DNA oligomer (ssO) used as a C-NHEJ competitor in the pull-down experiments is as follows: 5′-GTGTGAGTGTGAGTGTGAGTGTGAGTGTGAGTGTG-3′. 2.4. In vitro pull-down experiment Whole cell extracts were prepared as previously described 21 . The streptavidin magnetic beads (Beaver, China) were washed twice with Buffer Ⅰ (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 M NaCl, 0.01–0.1% Tween-20). Approximately 80 pmol of 75 bp biotinylated DNA probe was immobilized on 20 µl of beads in Buffer Ⅱ (0.1% Tween-20, phosphate-buffered saline (PBS) pH 7.4, 0.01–0.1% bovine serum albumin (BSA), as recommended by the manufacturer. Sixty micrograms of NHEJ-competent extract was incubated with 10 mM glucose and 1 U hexokinase (Solarbio, China) at 20°C for 5 min, and then 100 pmol of ssO was added and incubated at 4°C for 10 min. Ten microliters of mock- or 75 bp biotinylated DNA-treated beads was added to the extracts, washed with 0.5 × PBS, and incubated with a final 10 µl of Buffer Ⅲ (50 mM triethanolamine pH 8.0, 40 mM potassium chloride, 0.5 mM magnesium acetate, 1 mM dithiothreitol (DTT), 0.1 mg/ml BSA ) at 10°C for 25 min under mild agitation. The magnetic beads were washed with 0.5 × PBS and then used for western blotting to detect DNA-PKcs, Ku80, Ku70, Lig4, Lig1, and PARP-1. 2.5. Mass spectrometry The magnetic beads with bound biotinylated DNA fragment were incubated with whole cell extracts treated with ssO. The procedure used was similar to that used in the “ In vitro pull-down experiment.” The pull-down complex proteins bound to the beads were analyzed by mass spectrometry 22 . 2.6. Reverse transcription quantitative PCR (RT-qPCR) According to the manufacturer’s protocol, total RNA was extracted by RNAiso Plus (Takara). 1 µg of total RNA was reverse transcribed in a 20 µl reaction at 37°C using PrimeScript TM RT reagent Kit (Takara). According to the manufacturer’s instruction, RT-qPCR was carried out in MiniOpticon (Thermo Fisher Scientific, Waltham, MA, USA) with TB Green TM Premix Ex Taq TM Ⅱ (Takara). ddH 2 O, primers and 1 µl cDNA were prepared as described above. Primers were shown in Supplementary Table S1 . Samples of cDNA were heated to 95°C for 3 min followed by 40 cycles of 94°C 15 s, 57°C 30s, 72°C 20 s. The expression of different genes was analyzed by 2 − ΔΔCT. All PCR analyses were performed in triplicates. 2.7. Chromatin fractionation and immunoblotting Chromatin fractions Ⅰ-Ⅳ were isolated as previously described 21 . Cells were treated with or without Cali, washed with PBS, and harvested. Briefly, 200 µl of buffer Ⅰ (1 mM EDTA, 150 mM NaCl, 50 mM HEPES, pH 7.5, 0.05% Nonidet P-40 (NP40), with phosphatase and protease inhibitors (Solarbio, China) was added to the cells and incubated for 5 min on ice. The supernatant (fraction I) was collected after centrifugation at 1000 × g for 5 min; 200 µl of buffer Ⅰ containing 100 µg/ml RNase A was added to the pellet and incubated at 20°C for 10 min. The supernatant was collected as before (fraction II). Buffer Ⅰ containing 0.5% NP40 (200 µl) was again added to the nuclear pellets and incubated on ice for 40 min. The supernatant was collected after centrifugation at 16000 × g for 15 min (fraction Ⅲ). Two hundred microliters of buffer Ⅰ supplemented with 1% Triton X-100 and 0.45 M NaCl was added to the pellets and sonicated (fraction IV). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was then added to chromatin fractions I–IV. The fractions were boiled and then processed for western blotting. 2.8. Transfections of short interfering RNAs (siRNAs) and transduction All siRNAs employed were from GenePharma (Shanghai, China) and the sequence of EXO1 targeted by the siRNAs is as follows: EXO1#-1: 5′-CCATGTGAGGAAGTATAAA-3′, EXO1#-2: 5′-CCAATCTTCTTAAGGGAAA-3′. For siRNA transfection, the cells were plated in 6-well plates overnight and then transfected with siRNA targeting EXO1 using RFectPM transfection reagent (Changzhoubaidai, China). The adenovirus-shKu70 (ADV1, U6/CMV-GFP: 5′-CCAGTGTATCCAAAGTGTGTA-3′) was purchased from GenePharma (Shanghai, China). Cells were plated in 6-well plates overnight and then transducted with adenovirus-shKu70 for one to seven days. 2.9. DSB reporter assays The plasmids pimEJ5GFP (EJ5), EJ2GFP-puro (EJ2), and pCBASceI were purchased from Addgene (Cambridge, MA, USA; https://www.addgene.org/ ). A549 cell lines, each with an integrated reporter (EJ2 or EJ5), were generated according to a previously published integration protocol 23 . First, siRNA transfections were utilized in the DSB reporter assays: 5 × 10 4 A549-EJ2/EJ5 cells were seeded in a 24-well plate with 30 pmol of each siRNA mixed with 2 µl RfectPM transfection reagent (Changzhoubaidai, China) in 0.5 ml of medium without antibiotic. The cells were cultured for 24 h. Second, cells were transfected with 2 µl Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) mixed with 1 µg of I-SceI endonuclease expression vector (pCBASceI) in 0.5 ml antibiotic-free media. The cells were then cultured for 48 h. 2.10. Survival experiments For the survival experiments, A549, EXO1-depleted A549, A549/DDP, and EXO1-depleted A549/DDP cells (5 × 10 3 ) were individually seeded in 96-well plates. Various concentrations of DDP and / or DNA-PK inhibitor (NU7026) were added to the cells for 24 h. Cell viability was assayed using Cell Counting Kit-8 (CCK-8) (Solarbio, China) and a microplate reader. 2.11. Single-cell gel electrophoresis (SCGE) assay Cells were plated in 12-well plates at 1 × 10 5 cells/well. The cells were treated with 4 mg / l DDP for 6 h, 10 µM NU7026 for 24 h, or 10 µM NU7026 for 24 h followed by 4 mg / l DDP for 6 h and then analyzed by SCGE at different recovery times, according to published procedures 24 . Image analysis of at least 50 cells was performed using CometScore software. Data are expressed as means ± SEM (n = 50). 2.12. Western blot Cell line extracts were prepared by RIPA lysis buffer (Beyotime, China). SDS loading buffer was added to cell extracts. 30 µg of cell extracts were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with indicated antibodies and detected with chemiluminescent immunoassay (baoguang, China). The following antibodies were used: anti-RBMX (ab190352, Abcam; Cambridge, United Kingdom), anti-EXO1 (16253-1-AP, proteintech; Rosemont, USA), anti-DNA-PKcs (ab133516, Abcam; Cambridge, United Kingdom), anti-Ku70 (ab92450, Abcam; Cambridge, United Kingdom), anti-Ku80 (ab80592, Abcam; Cambridge, United Kingdom), anti-Lig4 (ab193353, Abcam; Cambridge, United Kingdom), anti-Lig1 (ab177946, Abcam; Cambridge, United Kingdom), anti-Lig3 (26893-1-AP, proteintech; Rosemont, USA), anti-PARP-1 (#9532, CST; Danvers, MA, USA), anti-caspase-3 (#9662, CST; Danvers, MA, USA), anti-PCNA(GTX100539, GeneTex; USA), anti-FEN1(GTX101777,GeneTex; USA)anti-ACTIN (66009-1-Ig, proteintech; Rosemont, USA). 2.13. Co-immunoprecipitation (Co-IP) Cells were washed with ice-cold PBS and then lysed in IP buffer (Beyotime, China) supplemented protease inhibitors for 30 min on ice. Cell lysates were removed by centrifugation and the supernatants were incubated with EXO1 antibody magnetic beads (MedChemExpress) overnight at 4°C. Magnetic beads were washed four times with 1 ml of PBST (1×PBS + 0.5% Triton X-100, pH 7.4). Bound proteins were eluted with 20–30 µL 2 × SDS-PAGE Loading Buffer and subjected to western blot analysis. 2.14. Immunofluorescence After fixation with 4% paraformaldehyde, the cells were permeabilized with PBS containing 0.1% Triton X-100. The cells were stained with 53BP1-, DNA-PKcs-, Ku70-, Ku80-, Lig4-, Lig1-, Lig3-, or PARP-1-specific antibody, washed twice, and then stained with goat anti-rabbit antibody F(ab´)2 fragment conjugated with Alexa Fluor 488 (Carlsbad, California, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Carlsbad, California, USA). 2.15. In vivo studies Animal experiments were approved by the Ethics Committee of Chongqing Medical University. Twenty, 5-week-old male Nu/Nu mice were obtained from Beijing Hua Yikang Biotechnology Co. Ltd. The mice were randomly assigned to four groups (five mice per group). Cells (5 × 10 6 ) in 0.2 mL PBS were injected subcutaneously in the groin of each mouse. The volume of the tumor was measured every three days and recorded. DDP and EXO1 inhibitor (EXO1-i) were administered intraperitoneally daily for 3 weeks (Groups: PBS, 3 mg/kg DDP, 10 mg/kg EXO1-i, and 3 mg/kg DDP plus 10 mg/kg EXO1-i) when each tumor reached 0.5 cm in diameter. Tumor volumes were calculated as follows: volume = length × width × width × 0.5. After 4 weeks, all mice were euthanized, and the subcutaneous tumors were weighed. 2.16. Statistical analysis Differences between the indicated experimental groups were evaluated by one-way ANOVA using SPSS 11.5 software. P < 0.05 was considered statistically significant. 3. Results 3.1. Screening of candidate proteins involved in Alt-EJ pathway in vitro Our recent study showed that ssO can hijack Ku protein and result in stimulating error-prone Alt-EJ repair of DSBs, since Ku is the core protein to initiate C-NHEJ and to recruit the following repair proteins 17 . Based on this instant in vitro Alt-EJ pathway model, to acquire the damage repair related protein pool in Alt-EJ process, we performed a pull-down experiment to harvest proteins recruited to DNA ends for further identification. One end of the dsDNA probe is biotinylated and bound to streptavidin-coated magnetic beads 25 . After the introduction of ssO (competitor DNA), Ku protein was bound to the ssO and dislocated from dsDNA in vitro 17 , the proteins bound at dsDNA ends contains DSBs are harvested and identified. In the absence of ssO, we found that Lig4 and DNA-PK (DNA-PKcs and Ku) were specifically bound to the dsDNA probes, clearly separated from the soluble proteins. When most Ku was attached to ssO in soluble extract, PARP-1 was found to bind to the dsDNA probes, demonstrating the competition between the Ku and PARP-1 in binding to dsDNA ends, which is consistence with previous literatures 21 , 26 . However, PARP-1 was also recovered on dsDNA probes in the absence of ssO, probably due to excess, non-limiting dsDNA in the assay. Under our conditions, Lig1 involved in the Alt-EJ pathway was not recruited to dsDNA beads (Fig. 1 A). In Fig. 1 B, the molecular weight of the band marked by the red arrow in Fig. 1 B is the same as that of DNA-PKcs, Ku80, and Ku70, respectively. The results in Fig. 1 B are similar to those in Fig. 1 A. These biochemical results have shown different binding affinity of main known C-NHEJ and Alt-EJ proteins in the presence or absence of ssO. The binding of Ku with ssO prevents the binding of DNA-PK and Lig4-dependent protein complexes with the dsDNA ends, which allows PARP-1 recruitment at dsDNA ends in lung cancer cell extract. Based on this system, to explore unidentified repair proteins involved in the Alt-EJ pathway, we utilized mass spectrometry to screen the proteins pool recovered on the dsDNA probe in the presence of ssO. Using the bioinformatics Gene Ontology (GO) enrichment method to analyze mass spectrometry data, we obtained 8 enrichments containing 21 genes related to DNA damage repair (Fig. 1 C). To identify highly expressed genes in lung cancer cells, the expression of 21 genes in HBE, H520, A549, and A549/DDP cells was quantified by RT-qPCR and further compared with western blot. The results of RT-qPCR showed that RNA binding motif protein X-linked (RBMX) and EXO1 were expressed higher in A549 and A549/DDP cells than that in normal bronchial epithelium HBE cells (Fig. 1 D, 1 E, 1 F, 1 G). In this study, Ribosomal protein S27 (RPS27), far upstream element-bingding protein 1 (FUBP1) and transcription elongation factor S-II (TCEA1) were also expressed higher in A549, A549/DDP, or H520 cells. Studies have shown that RPS27, FUBP1, and TCEA1 proteins are involved in DNA damage repair 27 – 29 , however, there is no clear research showing that these proteins take participate in DSB repair. POLQ and FEN1 have been identified to be involved in Alt-EJ repair 6 , 30 . Nimonkar et al. showed evidence that EXO1 may participate in the initial end resection step of microhomology-mediated end joining (MMEJ) 9 , 31 . Since Alt-EJ events are characterized as microhomology, which require end resection and join the ends at microhomology sequences. The activity of exonuclease is needed in this process. To prove EXO1 participating in Alt-EJ pathway, more detailed experiments need to carry out as below. Apart from candidate EXO1, in addition, Adamson et al. indicated that RBMX participates in HR, but in a PARP-1-dependent manner, suggesting that RBMX may also be a candidate protein in the Alt-EJ pathway 32 . The western blot results showed that the expression of EXO1 were higher than that in normal cells, which is consistent with the RT-qPCR results (Fig. 1 H, 1 I) .According to the RT-qPCR results and reported studies, we selected RBMX and EXO1 for subsequent investigation. 3.2. Recruitment of EXO1 to chromatin in response to radiomimetics in Ku70-deficient lung cancer cells To observe the distribution and colocation of C-NHEJ and Alt-EJ pathway proteins in cells, before and after Cali treatment, we performed immunofluorescence technique. We found that 53BP1, DNA-PKcs, Ku70, Ku80, Lig4, Lig1, Lig3, and PARP-1 proteins were localized to the nucleus, where they were uniformly distributed (Fig. 2 A). After treatment with Cali to induce DSBs, repair proteins aggregated around the broken chromatin (Fig. 2 A). These results suggest that proteins involved in DNA damage repair are recruited to the ends of DSBs of the broken chromatin. To further verify the binding affinity of EXO1 and RBMX to chromatin after radiomimetic treatment in lung cancer cells, we performed fractionation method. This protocol adopts continuous detergent extractions (fractions I–IV, representing cytoplasm to chromatin) which gradually removing loosely bound proteins from the chromatin, allowing visualization of the association of repair proteins at DSB sites in the chromatin. In Cali treated A549 and A549/DDP cells, as expected, most key proteins of C-NHEJ pathway were released in the early extracted I and II, retained in extraction resistant fractions III and IV (Fig. 2 B, 2 C). A marker of DSBs, γ-H2AX, was detected in insoluble fraction IV. In contract, Alt-EJ proteins PARP-1, Lig-1 and Lig-3 remained mainly in extractable fractions, with or without Cali treatment, displaying their low affinity for chromatin under this condition. With a similar distribution pattern with Alt-EJ proteins, EXO1 was consistent found in fraction I and II. However, nuclear protein RBMX was distributed differently from the known C-NHEJ and Alt-EJ pathway proteins. It possesses notably constant high affinity with chromatin before or after Cali (Fig. 2 B, 2 C). We planned to investigate the distribution of proteins in the IV fractions after Cali treatment in Ku70-deficient and Ku70-normal cells. To further confirm whether the proteins in the Alt-EJ and C-NHEJ pathways differ in their ability to bind to chromatin when DSBs occur, we used adenovirus-shKu70 to knockdown Ku70 expression by transducing A549 cells. We confirmed high infection efficiency by microscopy (Supplementary Fig. S1 ). Infected cells were harvested and subjected to western blots for Ku70 expression after five days. Expression of Ku70 proteins was reduced (Fig. 3 A). We then observed the known and candidate repair proteins localization with immunofluorescence and distribution in the four fractions in the Ku70-deficient A549 cells treated with or without Cali for 2 h (Fig. 3 B, 3 C, 3 D). In immunofluorescence experiment, DNA-PKcs, Ku80, and Lig4 distributed from the nucleus to the cytoplasm after Ku70 knockdown, while Lig1 and Lig3 partially remained in the nucleus in the Cali treated group, suggesting that these proteins undergo redistribution in the cells (Fig. 3 B). To observe the binding affinity the proteins, fractionation method was performed as before. In the insoluble fractions, γ-H2AX was present. In A549 cells, in the presence of Ku70, Ku and Lig4 were detected in fractions III and IV (Fig. 2 B 2 C, Figure. 3C) after Cali treatment. PARP-1 was mainly distributed in soluble fractions (I and II), and some in the less soluble fraction III, along with Lig-1 and Lig-3, probably due to the presence of SSBs in DNA in the system. With Ku70 deficiency, PARP-1 shifted to the insoluble fraction IV, indicating their increased chromatin binding affinity (Fig. 3 C). These data clearly showed a shift from C-NHEJ to Alt-EJ when Ku70 expression was limited in A549 cells. We verified whether RBMX and EXO1 participate in the chromatin binding process as those known C-NHEJ or Alt-EJ proteins. Notably, the results showed that the distribution of EXO1 was similar to that of the known Alt-EJ pathway proteins. In Ku70 deficiency condition, EXO1 can be detected accumulated in the insoluble fraction IV. RBMX’s distribution was different (Fig. 3 C). Similar results were confirmed in A549/DDP cells (Fig. 3 D). These data showed EXO1 binding to the DSB ends under Ku70 deficiency condition, preliminarily indicating that EXO1 may be participate in the Alt-EJ pathway, whereas the recruitment patter of RBMX fail to demonstrate its involvement in Alt-EJ. 3.3. EXO1 is involved in the mechanism of Alt-EJ After demonstrating the recruitment of EXO1 to the radiomimetic-induced DSB, to further explore whether it participates in the DSB repair mechanism and contributes to the ligation effect, we used two chromosome substrates that allowed DSBs to be introduced into. The two reporter substrates were EJ5-GFP 18 – 20 which indicates the repair of C-NHEJ, and EJ2-GFP 33 , 34 , indicates that of Alt-EJ (Supplementary Fig. S2 A, S2B). The cells were transfected with pCBASceI to induce DSBs at the I-SceI site of the reporter plasmid. The percentages of GFP + cells reflected the rate of C-NHEJ or Alt-EJ in the cells, respectively. A549 cells were transfected with negative control siRNA, siRNA-1 targeting EXO1, or siRNA-2 targeting EXO1. Western blot confirmed that the expression of EXO1 was lower in cells transfected with siRNA-1 or siRNA-2 compared to cells transfected with the negative control siRNA (Supplementary Fig. S2 C). In parallel, siRNAs targeting EXO1 were transfected into A549-EJ5 cells and A549-EJ2 cells, followed by 24 h incubation to ensure downregulation of EXO1. The flow cytometry results for GFP + cells are shown in Fig. 4 A and the percentage of GFP + cells are quantified in Fig. 4 B. Compared to the negative control siRNA-transfected cells, the cells depleted of EXO1 with siRNA-1 exhibited a significantly decreased percentage of EJ2-GFP. Importantly, the level of GFP expressed in A549-EJ2 cells depleted of EXO1 by two different siRNAs, which indicates more suppression of EXO1 expression, was significantly lower than that in cells depleted of EXO1 by one siRNA (Fig. 4 B). In these data, we can conclude that the inhibition of EXO1 compromises the repair of DSB with Alt-EJ pathway in cancer cells, indicating EXO1’s contribution in Alt-EJ ligation. Notably, we found that the percentage of EJ5-GFP + cells was significantly different between the control and siRNA-treated cells (Fig. 4 A, 4 B), suggesting that depletion of EXO1 may negatively regulate the C-NHEJ pathway. To investigate whether inhibiting of C-NHEJ would increase Alt-EJ in the presence or absence of EXO1, we used DNA-PK inhibitor NU7026 to treat cells and inhibit the C-NHEJ ligation of substrate. In cells pre-treated with NU7026, we found an increase in Alt-EJ ligation of substrate in the control cells (Fig. 4 C, 4 D). Suppression of C-NHEJ leads to activation of Alt-EJ, which was consistence with previous findings. In contrast, in cells pre-treated with NU7026 and transfected with EXO1 siRNA, the ligation quantity of EJ2-GFP substrate was much lower, compared to the NU7026 treated cells without EXO1 siRNA (Fig. 4 C, 4 D). The results showed that in DNA-PK suppression condition, inhibition of EXO1 compromise the C-NHEJ independent ligation, demonstrating that when the C-NHEJ pathway is inhibited, the Alt-EJ pathway relies, at least in part, on EXO1 to repair DSB in lung cancer cells. Altogether, our results exhibit EXO1 participation in DSB repair mechanism of Alt-EJ. Research showed that proliferating cell nuclear antigen (PCNA) loads onto double-strand breaks and directly interacts with EXO1 35 . FEN1, Alt-EJ pathway protein, strongly interacts with PCNA to promote DNA repair 36 . PCNA interacts with FEN1 and Lig1 to complete Okazaki fragment processing and joining 37 . Therefore, we speculated that EXO1 may participate in Alt-EJ via interacting with proteins in the Alt-EJ pathway. In order to further confirm this conjecture, we performed the Co-IP experiment. Our results showed that EXO1 interacted with PCNA, FEN1, and Lig1, but not with Ku70 (Fig. 4 D). These results further confirm that EXO1 participates in the Alt-EJ pathway by combining with FEN1 and Lig1 involved in the Alt-EJ pathway. 3.4. EXO1-depleted cancer cells are more sensitive to DDP Expression of EXO1 is associated with the therapeutic effect of drugs that induce DSBs in tumor cells 16 . To clarify the relationship between EXO1 and DDP resistance, we used siRNA to knockdown EXO1 in A549/DDP and A549 cells and then treated them with DDP. Cell survival percentage was observed and evaluated. siRNA-mediated depletion of EXO1 led to an overall higher sensitivity of A549 cells to DDP than A549 cells treated with negative control siRNA at each DDP concentration (Fig. 5 A). Similar results were observed in A549/DDP cells (Fig. 5 B). Inhibition of DNA-PK with NU7026 to impede C-NHEJ led to increased levels of Alt-EJ in cells treated with negative control siRNA compared to that in A549-EJ2 cells treated with EXO1-targeted siRNA. This suggests that at least a portion of the DSB ends were shunted to be repaired by Alt-EJ in our cells, according to our reporter plasmid system results. Therefore, we investigated whether the depletion of EXO1 sensitizes A549 and A549/DDP cells to NU7026. A549 and A549/DDP cells were transfected with siRNA against EXO1 and then different concentrations of NU7026 were added. Compared to that of mock-depleted cells, NU7026 pretreated cells exhibited a significant decrease in viability in A549/DDP cells depleted of EXO1 (Fig. 5 C, 5 D). Next, we observed the sensitivity to DDP in A549, EXO1-depleted A549, NU7026-pretreated A549, and NU7026-pretreated EXO1-depleted A549 cells. As expected, the highest sensitivity to DDP was observed in NU7026 pretreated EXO1-depleted A549 cells (Fig. 5 E, 5 F). These results indicate that DDP-induced DNA damage is repaired by the EXO1-associated pathway when NU7026 inhibits the C-NHEJ pathway. EXO1-depleted cancer cells are more sensitive to DDP. 3.5. DSBs accumulate in EXO1-depleted cells Given that EXO1-depleted cancer cells are more sensitive to DDP, and this sensitivity can be increased by inhibition of C-NHEJ with NU7026 treatment (Fig. 5 E, 5 F), we reasoned that the elevated sensitivity might be related with increased levels of DSBs formation at DNA ends. To confirm this, we measured levels of DSBs using an SCGE assay with wild type (WT) and EXO1-depleted A549 and A549/DDP cells treated with DDP. We observed that the levels of DSBs continued to be significantly higher in the EXO1-depleted cells than in the WT cells of A549 and A549/DDP, even after recovering for 24 h (Fig. 6 B, 6 C). Representative images for each recovery time point after DDP treatment of A549 and A549 EXO1-depleted cells are shown in Fig. 6 A. We also found that compared to that in A549 and A549/DDP WT cells treated with the DNA-PK inhibitor NU7026 for 24 h, the levels of DSBs continued to be slightly but significantly higher in the EXO1-depleted A549 and A549/DPP cells (Fig. 6 D, 6 E). Furthermore, we observed that the EXO1-depleted cells showed significantly greater persistence of DSBs when they were treated with DDP and NU7026, compared with control groups (Fig. 6 F, 6 G). These results demonstrated that inhibition of EXO1 chemosensitizes cancer cells through promotes the formation of unrepaired DSBs after DDP treatment, especially when C-NHEJ was pharmacologically inhibited. 3.6. Depletion of EXO1 inhibits DDP-resistant tumor growth in vivo Based on the in vitro EXO1 inhibition, we examined the effect of the combined treatment of DDP and EXO1-i on an established xenograft model in vivo . DDP and EXO1-i combination treatment remarkably inhibited tumor growth, consistent with the in vitr o data (Fig. 7 A). Images of all tumors are presented in Fig. 7 B. A comparison of tumor volumes in the groups revealed that DDP and EXO1-i combination treatment substantially reduced tumor volumes, in agreement with the tumor weight results (Fig. 7 C, 7 D). As shown in Fig. 6 E, expression of apoptotic protein caspase-3 and 53BP1 was greatly increased in the combination therapy group when compared to that in the two other groups, as assessed by immunohistochemistry. All these data are consistent with the in vitro data and further confirm the synergistic anticancer effect of DDP and EXO1-i in vivo . 4. Discussion Yuan et al. was the first to describe the unexpected property of ssO, which activate Alt-EJ by hijacking Ku protein in the C-NHEJ pathway from DSB ends to make access for Alt-EJ proteins to DNA ends subsequently. The mechanism was extensively demonstrated to verify that it was established as an highly efficient Alt-EJ repair pathway system both in vitro and in cells 17 . In this study, in lung cancer cells, our pull-down experiments suggested that ssO could prevent Ku from binding to DSB ends while PARP-1 protein in the Alt-EJ pathway was recruited, which is consistent with the author’s previous work. We prove the mechanism can be widely applied in multiple cell types to achieve an efficient and convenient Alt-EJ repair pathway shift. Based on the mechanism, EXO1 was identified by mass spectrometry among proteins recruited to the magnetic beads in the presence of ssO, along with other known Alt-EJ proteins as FEN1 and POLQ. In our study, EXO1 was selected as a potential player involvement in Ku-independent Alt-EJ pathway in lung cancer cells. In cellular chromatin fractionation assays, we found the mobilization of EXO1 to insoluble fractions, indicating its recruitment to DNA ends under in vitro condition. We also observed that cells treated with Cali showed significantly increased Ku level and accumulated Lig4 in fraction IV, as well as accumulated Lig1, Lig3, and PARP-1 in fraction Ⅲ. With cell extracts, we further showed that Ku depletion in cells induced a shift from C-NHEJ to Alt-EJ. Using a previously published reporter plasmid assay to design to differentiate DSB ligation with C-NHEJ or microhomology based Alt-EJ 18–20 , we found that, in EXO1-depleted lung cancer cells, DSB repair with Alt-EJ was attenuated whereas C-NHEJ was enhanced. After treatment with a DNA-PK inhibitor, Alt-EJ increased, indicating that the cells were more dependent on Alt-EJ when C-NHEJ was inhibited. Importantly, we found that inhibition of C-NHEJ fail to enhance Alt-EJ in EXO1-depleted cells, indicating that EXO1 is associated with NHEJ independent microhomology dependent pathway in lung cancer cells. EXO1 contributes to checkpoint progression and to several DNA repair pathways involved in MMR, translesion DNA synthesis, NER, DSBs repair. By matched whole-genome sequencing data for an extensive study of cancer genome alterations, EXO1 is considered a new cancer-driver genes candidate and associated with survival in colorectal adenocarcinoma 38 . Exo1 null/null mice show an increase in chromosomal breaks and base substitution, and predominately develop lymphomas 39 , and cells depleted EXO1 also show chromosomal instability and hypersensitivity to ionizing radiation 40 . EXO1’s hyper-resection can attenuate both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. DNA-resection by EXO1 is probably inhibited by the DNA binders RPA, Ku70/80, and/or CtIP 41 , 42 . In C-NHEJ, the Ku70/80 heterodimer protects the DNA in a complex with DNA-PKcs for DNA end resection 43 . Therefore, EXO1 has a limited role in this pathway. In contrast, EXO1 likely collaborates in an Alt-EJ pathway with the WRN in trimming the DNA ends 44 . PARP-1, a factor involved in DSB Alt-EJ repair, physically interacts with EXO1 and stimulates EXO1 in its 5’ excision activity in an in vitro condition 45 . In liver cancer, EXO1 is significantly upregulated in HCC tumor tissues and that high EXO1 plays a carcinogenic role 46 . Elevated EXO1 mRNA and proteins expression was detected in lung cancer cells in our study. In ovarian cancer, EXO1 contributed to drug resistance 47 . In this study, we found that depletion of EXO1 in A549 and A549/DDP cells made them sensitive to DNA-PK inhibitor or DDP, which is related to the presence of persistent DSBs in these cells that were evaluated in SCGE analysis. Importantly, our data suggest that the state of EXO1 can influence the response of tumors to DDP. Based on our in vitro results, we examined the in vivo antitumor effect of DDP and EXO1-i treatment alone or in combination using an established xenograft model. Combination therapy with DDP and EXO1-i significantly increased tumor inhibition rates. 5. Conclusion In summary, we have demonstrated the role of EXO1 in Alt-EJ and found that the former contribute to DDP resistance. However, in these cases, the detailed mechanism of EXO1 end excision is still poorly understood. Genetic mutations in excision factors are associated with multiple genetic diseases, predisposition to cancer, and premature aging. Our study may also provide a potential target for enhancing the therapeutic efficacy of chemotherapy by the inhibition of EXO1. Declarations Authors' contributions : Ying Yuan conceived the idea of experiments. Yonghong Wang and Yalin Zhu performed the experiments. Hui Zhang interpreted and analyzed the data. Yonghong Wang wrote the draft of manuscript. Wenli Feng, Ying Yuan and Zhenglan Huang critically revised the manuscript. All authors read and approved the final manuscript. Funding: This work was supported by the National Natural Science Foundation of China (No.81703095) and the Innovation Support Program for Overseas Students of Chongqing (cx2018142). Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Ethics approval and consent to participate: Informed consent was obtained from all subjects involved in the study. 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1","display":"","copyAsset":false,"role":"figure","size":5905584,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ssO on repair protein enrichment at DNA ends in C-NHEJ and Alt-EJ pathway\u003c/p\u003e\n\u003cp\u003e(A) Streptavidin beads loaded with or without dsDNA probes were mixed with A549 cell extracts incubated or not incubated with ssO competitor. The amounts of dsDNA probes and ssO in the reaction were 80 pmol and 100 pmol, respectively. In pull-down experiments, a portion of the initial amount of protein is used as an input group. The proteins remaining insoluble or bound to the beads were subjected to SDS-PAGE and western blot analysis. (B) The procedure is the same as A. Samples were loaded on an SDS-PAGE gel, and then directly stained with Coomassie Brilliant Blue. M denotes protein marker. (C) DAVID website was used to analyze mass spectrometry data, which was drawn with R software. (D, E, F, G) The expression of 21 genes was determined by RT-qPCR in HBE, H520, A549, and A549/DDP cells. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. (H and I) Western blot was used to analyze the expression of RBMX and EXO1 in HBE, H520, A549, and A549/DDP cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01***, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/5927f3de484762fb614b199d.png"},{"id":100004401,"identity":"4c37d674-5c44-45bc-b418-99f3dc0610fa","added_by":"auto","created_at":"2026-01-12 05:26:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12840388,"visible":true,"origin":"","legend":"\u003cp\u003eNuclear localization and mobilization to damaged chromatin of repair proteins in C-NHEJ and Alt-EJ pathways\u003c/p\u003e\n\u003cp\u003e(A) A549 cells were treated with or without 40 nM Cali for 2 h and were then fixed, permeabilized, and immunostained with anti-53BP1, anti-Ku70, anti-Ku80, anti-Lig4, anti-Lig1, anti-Lig3, or anti-PARP-1 antibodies. Cell nuclei were stained with DAPI. A549 (B) and A549/DDP (C) cells treated (T) or not (NT) with 40 nM Cali for 2 h were harvested and subjected to fractionation to obtain fractions Ⅰ to Ⅳ as described in the “Chromatin fractionation and immunoblotting” section of the Material and Methods. Protein samples, Fraction Ⅰ, Fraction Ⅱ, Fraction Ⅲ, and Fraction Ⅳ, were denatured and separated by SDS-PAGE followed by electrophoretic transfer and western blotting as indicated.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/833d4c31b635b5fefd7b0597.png"},{"id":100004407,"identity":"8fc2cd8a-6efc-473d-b711-722128426056","added_by":"auto","created_at":"2026-01-12 05:26:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10879282,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of depleted Ku on nuclear localization and mobilization to damaged chromatin of repair proteins\u003c/p\u003e\n\u003cp\u003e(A) A549 cells were transduced with ADV1-Ku70-192 virus for one to seven days. Expression of Ku70 was detected by western blot. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. (B) A549 cells were transduced with ADV1-Ku70-192 for 5 days, and then treated with or without 40 nM Cali for 2 h. Then, cells were examined as in Figure 2A. A549 (C) and A549/DDP (D) cells were transduced with ADV1-shKu70 (+) or mock-ADV1 (-) for 5 days, and then treated with 40 nM Cali for 2 h, fractionated, and examined as in Figure 2B.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/89aad582237093fcb86a98a4.png"},{"id":100004399,"identity":"97462e65-9870-4b15-bee4-c938816f87cf","added_by":"auto","created_at":"2026-01-12 05:26:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2853590,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of depleted EXO1 on GFP reporters\u003c/p\u003e\n\u003cp\u003e(A and B) Stable A549 cell lines were established with EJ2-GFP or EJ5-GFP reporter. The stable cell lines were either treated with negative siRNA and pCBASceI or treated with siRNA targeting EXO1 and pCBASceI, and analyzed after 2 days. The percentage of GFP+ cells in the A549 EJ5/EJ2 cells was quantified by flow cytometry.Control, siRNA-1, and siRNA-1+siRNA-2 denote cells treated with negative control siRNA, siRNA-1, and siRNA-1/2 targeting EXO1, respectively. Data are means ± SD of triplicates. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. (C and D) A549-EJ2 cells were treated with negative control siRNA, siRNA that targets EXO1, or NU7026, an inhibitor of DNA-PK. The percentage of GFP+ cells was detected by flow cytometry. Data are means ± SD of triplicates. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. (E) Co-immunoprecipitation of lysates from A549 untreated (-) or treated with (+) Cali (44 nM) for 1 h. Western blots demonstrate association of indicated proteins.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/101f387e0a31c6f59b018239.png"},{"id":100004427,"identity":"73e2ef16-9a2e-4eef-98b4-85507712757d","added_by":"auto","created_at":"2026-01-12 05:26:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1999858,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DDP or/and NU7026 on the proliferation of A549 and A549/DDP cells\u003c/p\u003e\n\u003cp\u003e(A) A549 and A549-EXO1-depleted cells were treated with a series of concentrations of DDP for 24 h. Data are presented as means ± SD of triplicates. *\u003cem\u003eP\u003c/em\u003e=0.05. (B) A549/DDP and A549/DDP-EXO1-depleted cells were treated with various concentrations of DDP for 24 h. Data are presented as means ± SD of triplicates. *\u003cem\u003eP\u003c/em\u003e=0.05. (C) A549 and A549-EXO1-depleted cells were treated with various concentrations of NU7026 for 24 h. Data are presented as means ± SD of triplicates. *\u003cem\u003eP\u003c/em\u003e=0.05. (D) A549/DDP and A549/DDP-EXO1-depleted cells were treated with various concentrations of NU7026 for 24 h. Data are presented as means ± SD of triplicates. *\u003cem\u003eP\u003c/em\u003e=0.05. (E) EXO1-depleted A549 and A549 cells were pretreated with 10 µM NU7026 for 24 h and then treated with a range of concentrations of DDP for 24 h. Data are presented as means ± SD of triplicates. *\u003cem\u003eP\u003c/em\u003e=0.05. (F) EXO1-depleted A549/DDP and A549/DDP cells were pretreated with 10 µM NU7026 for 24 h and then treated with a range of concentrations of DDP for 24 h. Data are presented as means ± SD of triplicates. *\u003cem\u003eP\u003c/em\u003e=0.05\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/cb5e9d634261e7964056e389.png"},{"id":100004432,"identity":"dfca1ef4-4227-438a-b841-aa492a5e0540","added_by":"auto","created_at":"2026-01-12 05:26:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6536364,"visible":true,"origin":"","legend":"\u003cp\u003eEXO1-depleted cells have increased levels of DSBs upon treatment with DDP or NU7026\u003c/p\u003e\n\u003cp\u003e(A, B and C) DDP was added to A549 cells (A, B), A549-EXO1-depleted cells (A, B), A549/DDP cells (A, C), and A549/DDP-EXO1-depleted cells (A, C) at 4 mg/l for 6 h and the cells were allowed to recover for 12 h or 24 h. DSBs were measured by SCGE assay. Data are presented as means ± SEM. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. The tail moment was analyzed by CometScore software. (D and E) 10 µM NU7026 was added to A549 cells (D), A549-EXO1-depleted cells (D), A549/DDP cells (E), and A549/DDP-EXO1-depleted cells (E) for 24 h. DSBs detection and analysis are the same as in part A. Data are presented as means ± SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05. (F and G) 10 µM NU7026 was added to A549 cells (F), A549-EXO1-depleted cells (F), A549/DDP cells (G), and A549/DDP-EXO1-depleted cells (G) for 24 h, 4 mg/l DDP was added for 6 h, and the cells were allowed to recover for 12 h or 24 h. DSBs detection and analysis are the same as in part A. Data are presented as means ± SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/5e69d7f35e7ae2371e0da86b.png"},{"id":100361297,"identity":"9ba42884-47c8-4e20-a9c1-82896667ad0e","added_by":"auto","created_at":"2026-01-16 07:44:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":22735679,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of EXO1 increases chemosensitivity to DDP \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Xenograft models were established by subcutaneous injection of 5 × 10\u003csup\u003e6\u003c/sup\u003e A549/DDP cells into the groin of mice. (B) Image of solid tumors obtained from different groups. Tumor volumes from the mice (C), and tumor weights (D) from the mice. Data are shown as means ± SD. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (E). Immunostaining assays to detect changes in levels of caspase-3 and 53BP1 (400×).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/ad416dac33616e42a5e1c73e.png"},{"id":102766367,"identity":"54c829f2-574c-41e5-b419-5c2433fab25d","added_by":"auto","created_at":"2026-02-16 11:27:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":60354401,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/e0367a38-0c0f-4f40-9a4c-061243439f6b.pdf"},{"id":100004395,"identity":"922210ff-3af5-40ef-b9ee-6c623c7cddbb","added_by":"auto","created_at":"2026-01-12 05:26:00","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7717896,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/084a488490a9e1e7cf2a3835.tif"},{"id":100004396,"identity":"901e7a78-b830-4bdf-ac08-bba8b0405011","added_by":"auto","created_at":"2026-01-12 05:26:00","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8775244,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/bfee4a4172e9cbe8eeaba2b1.tif"},{"id":100360306,"identity":"c0089f5f-94ba-48c0-af89-d000226cc1f1","added_by":"auto","created_at":"2026-01-16 07:38:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21565,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8486386/v1/15b0fa5b69433eb555940a54.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"EXO1 is associated with Ku-independent DNA double strand break repair pathway and the inhibition of EXO1 chemosensitizes DDP resistance lung cancer cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDouble-strand breaks (DSBs) are the most lethal type of DNA damage, which are induced by endogenous cellular processes, such as V(D)J recombination and oxidative metabolism, as well as exogenous agents like ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors. In mammalian cells, there are two major repair pathways for DSBs, homologous recombination (HR) and canonical non-homologous end joining (C-NHEJ). The former requires sister chromatin as the template to complete high-fidelity repair. The C-NHEJ operates through the whole cell cycle, and is initiated by the binding of Ku70/Ku80 heterodimer, followed by DNA-dependent protein kinase catalytic subunits (DNA-PKcs), DNA ligase 4 (Lig4), XRCC4, XRCC4-like factor (XLF) etc. Ku provides protection of DNA ends from degradation and recruits other repair proteins, which make Ku the most important protein in maintenance of C-NHEJ pathway.\u003c/p\u003e \u003cp\u003eWhile C-NHEJ is unavailable, especially in the absence of Ku from DNA ends, an error-prone alternative end joining (Alt-EJ) takes over as a backup option. It functions independent of nonhomologous end joining (NHEJ) or HR core components and is highly relevant to the chromosomal instability associated with cancer. In the past decades, proteins like poly (ADP-ribose) polymerase 1 (PARP-1), DNA Ligase 1 (Lig1), XRCC1/DNA Lig-3, C-terminal binding interaction protein (CtIP), DNA polymerase theta (POLQ) etc. have been identified participating in the pathway. More unrevealed proteins are on the way of being discovered. Whether Alt-EJ is an independent pathway remains controversial. Unlike other classical repair pathway, it forms a bricolage at DNA ends \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e by sharing players also involved in some other repair pathways, like PARP-1 in base-excision repair pathway for DNA single-strand breaks (SSBs) repair and MRN complex and CtIP of initial resection step of HR.\u003c/p\u003e \u003cp\u003eAnother feature of the Alt-EJ pathway is the formation of a high frequency of microhomologies and information deletions at ligated ends \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, which increases the likelihood of gene mutations and leads to chromosomal instability. Notably, apart from tumorigenesis, some evidence revealed that it might aide to generate and reinforce the resistance to anticancer drugs. For example, in chronic myeloid leukemia (CML), it\u0026rsquo;s a probable cause for BCR-ABL gene mutations that help to form the resistance to BCR-ABL inhibitors \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Moreover, the increased contribution of the error-prone Alt-EJ pathway to anticancer drugs resistance is at least partially due to increased expression levels of the known error-prone Alt-EJ factors. For instance, DNA Lig3 and PARP-1 display higher steady state levels in tyrosine kinase inhibitor-resistant CML \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and therapy-resistant breast cancer cell lines \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Importantly, survival of HR deficient tumors is promoted by mutagenic Alt-EJ DNA repair pathway \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Knockdown of POLQ is highly effective in killing HR-deficient cells, raising the possibility that cell proliferation in the absence of HR is dependent on Alt-EJ \u003csup\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn anticancer chemotherapy, one of major mechanisms of Cisplatin (DDP) resistance is DNA damage repair, due the highly efficient repair system and elevated DNA repair capacity after cell damage, which enables cancer cells exhibit intrinsic resistance and undermines the efficacy of drug cytotoxicity. The known relevant repair pathways include nucleotide excision repair (NER), interstrand cross-links (ICLs), mismatch repair(MMR)and HR. In some cell lines, like ovarian A2780 cells, the DNA repair mechanism is not the major cause of DDP resistance. In cell lines that are sensitive to DDP, the DNA repair ability is weaker than that in cells resistant to DDP \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDNA double-strand break repair by HR begins with nucleolytic resection of the 5' DNA strand at the break ends. The end resection comprises the generation of relative short stretches of ssDNA by MRN and CtIP and extensive resection by EXO1 and BLM-DNA2. The latter has been considered to favor HR \u003csup\u003e9\u003c/sup\u003e. However, Alt-EJ shares initial resection step with HR which uses DNA resection to reveal single-stranded MHs for annealing before joining. Resection proteins like MRN complex and CtIP are versatile and have been demonstrated participating in C-NHEJ, HR and Alt-EJ \u003csup\u003e10\u0026ndash;15\u003c/sup\u003e. Overexpression of EXO1 induces hyper-resection to attenuate both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The regulation of EXO1 is critical for accurate end joining and the mechanism of resection is far from fully understood, whether EXO1 participate in Alt-EJ worth an investigation.\u003c/p\u003e \u003cp\u003eGiven Ku is considered the most essential factor to keep Alt-EJ marginal and remain the a faithful repair, in our previous work, we established a novel Alt-EJ model in which Ku can be hijacked by short single-stranded oligonucleotides (ssO) generated from artificial interference or DNA end resection \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The preventing of Ku accumulation DSBs ends favors Alt-EJ and make instant activation of Alt-EJ possible. This discover shed a light into the possibility that Alt-EJ can be locally activated at some DNA ends where Ku is relatively lacks and eventually lead to the chromosomal instability and locally initiation of cancer. Based on this model, in this study, Alt-EJ was favored at ends of biotinylated probe dsDNA in the presence of ssO in cell extracts \u003cem\u003ein vitro\u003c/em\u003e. Damage repair related proteins participated in Alt-EJ was harvested in pull-down assay, screened with mass spectrometry analysis, and selected with RT-qPCR and western blotting. The chromatin binding affinity of candidate proteins was examined by biochemical fractionation and by immunostaining. EXO1, as one of our selected Alt-EJ protein, is found recruited to the DSB sites upon DNA damage in a C-NHEJ independent manner in Ku70-deficient lung cancer cells, in which Ku70 was down-regulated by using a ADV1-Ku70-192 virus to active Alt-EJ. Using a reporter plasmid assay in cells \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, Alt-EJ was found decreased and C-NHEJ was increased in EXO1-depleted cells. Evaluated in SCGE analysis, EXO1-depleted cells exhibited higher sensitivity to DDP or DNA-PK inhibitor by formation of more DSBs. In xenograft model, combination therapy with DDP and EXO1-i significantly increased tumor inhibition rates. In our condition, we found that, in Ku70-deficient cancer cells, the chromatin-binding affinity of EXO1 increased in response to DSBs in C-NHEJ independent manner, revealing its role in Alt-EJ-mediated DNA repair. By regulating the repair pathway, it promotes cell survival, mediates cell resistance to chemotherapy agent DDP and promotes DSBs formation at DNA end. Herein, EXO1 is a potential candidate for developing lung cancer-specific drugs and customized targeted molecular therapy.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents\u003c/h2\u003e \u003cp\u003eCalicheamicin-γ1 (Cali), purchased from MedChemExpress (Monmouth junction, NJ, USA), was dissolved in DMSO at 4 mM and stored at -80\u0026deg;C. NU7026 was also purchased from MedChemExpress (Monmouth junction, NJ, USA) and dissolved in DMSO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell culture\u003c/h2\u003e \u003cp\u003eHBE, H520, A549, and A549/DDP drug resistence cell lines were purchased from the China Center for Type Culture Collection (Wuhan, China) and were grown in Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA) containing penicillin-streptomycin and 10% fetal bovine serum (FBS, Gibco, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. DNA fragments and oligonucleotides\u003c/h2\u003e \u003cp\u003eThe sequence of the 75-bp DNA fragment used in pull-down experiments is as follows: 5\u0026prime;-CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC-3\u0026prime;, 5\u0026prime;-GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACG-3\u0026prime;. The sequence of the single-stranded DNA oligomer (ssO) used as a C-NHEJ competitor in the pull-down experiments is as follows: 5\u0026prime;-GTGTGAGTGTGAGTGTGAGTGTGAGTGTGAGTGTG-3\u0026prime;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. In vitro pull-down experiment\u003c/h2\u003e \u003cp\u003eWhole cell extracts were prepared as previously described \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The streptavidin magnetic beads (Beaver, China) were washed twice with Buffer Ⅰ (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 M NaCl, 0.01\u0026ndash;0.1% Tween-20). Approximately 80 pmol of 75 bp biotinylated DNA probe was immobilized on 20 \u0026micro;l of beads in Buffer Ⅱ (0.1% Tween-20, phosphate-buffered saline (PBS) pH 7.4, 0.01\u0026ndash;0.1% bovine serum albumin (BSA), as recommended by the manufacturer. Sixty micrograms of NHEJ-competent extract was incubated with 10 mM glucose and 1 U hexokinase (Solarbio, China) at 20\u0026deg;C for 5 min, and then 100 pmol of ssO was added and incubated at 4\u0026deg;C for 10 min. Ten microliters of mock- or 75 bp biotinylated DNA-treated beads was added to the extracts, washed with 0.5 \u0026times; PBS, and incubated with a final 10 \u0026micro;l of Buffer Ⅲ (50 mM triethanolamine pH 8.0, 40 mM potassium chloride, 0.5 mM magnesium acetate, 1 mM dithiothreitol (DTT), 0.1 mg/ml BSA ) at 10\u0026deg;C for 25 min under mild agitation. The magnetic beads were washed with 0.5 \u0026times; PBS and then used for western blotting to detect DNA-PKcs, Ku80, Ku70, Lig4, Lig1, and PARP-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Mass spectrometry\u003c/h2\u003e \u003cp\u003eThe magnetic beads with bound biotinylated DNA fragment were incubated with whole cell extracts treated with ssO. The procedure used was similar to that used in the \u0026ldquo;\u003cem\u003eIn vitro\u003c/em\u003e pull-down experiment.\u0026rdquo; The pull-down complex proteins bound to the beads were analyzed by mass spectrometry \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Reverse transcription quantitative PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer\u0026rsquo;s protocol, total RNA was extracted by RNAiso Plus (Takara). 1 \u0026micro;g of total RNA was reverse transcribed in a 20 \u0026micro;l reaction at 37\u0026deg;C using PrimeScript\u003csup\u003eTM\u003c/sup\u003eRT reagent Kit (Takara). According to the manufacturer\u0026rsquo;s instruction, RT-qPCR was carried out in MiniOpticon (Thermo Fisher Scientific, Waltham, MA, USA) with TB Green\u003csup\u003eTM\u003c/sup\u003ePremix Ex Taq\u003csup\u003eTM\u003c/sup\u003eⅡ (Takara). ddH\u003csub\u003e2\u003c/sub\u003eO, primers and 1 \u0026micro;l cDNA were prepared as described above. Primers were shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Samples of cDNA were heated to 95\u0026deg;C for 3 min followed by 40 cycles of 94\u0026deg;C 15 s, 57\u0026deg;C 30s, 72\u0026deg;C 20 s. The expression of different genes was analyzed by 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT. All PCR analyses were performed in triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Chromatin fractionation and immunoblotting\u003c/h2\u003e \u003cp\u003eChromatin fractions Ⅰ-Ⅳ were isolated as previously described \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Cells were treated with or without Cali, washed with PBS, and harvested. Briefly, 200 \u0026micro;l of buffer Ⅰ (1 mM EDTA, 150 mM NaCl, 50 mM HEPES, pH 7.5, 0.05% Nonidet P-40 (NP40), with phosphatase and protease inhibitors (Solarbio, China) was added to the cells and incubated for 5 min on ice. The supernatant (fraction I) was collected after centrifugation at 1000 \u0026times; g for 5 min; 200 \u0026micro;l of buffer Ⅰ containing 100 \u0026micro;g/ml RNase A was added to the pellet and incubated at 20\u0026deg;C for 10 min. The supernatant was collected as before (fraction II). Buffer Ⅰ containing 0.5% NP40 (200 \u0026micro;l) was again added to the nuclear pellets and incubated on ice for 40 min. The supernatant was collected after centrifugation at 16000 \u0026times; g for 15 min (fraction Ⅲ). Two hundred microliters of buffer Ⅰ supplemented with 1% Triton X-100 and 0.45 M NaCl was added to the pellets and sonicated (fraction IV). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was then added to chromatin fractions I\u0026ndash;IV. The fractions were boiled and then processed for western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Transfections of short interfering RNAs (siRNAs) and transduction\u003c/h2\u003e \u003cp\u003eAll siRNAs employed were from GenePharma (Shanghai, China) and the sequence of EXO1 targeted by the siRNAs is as follows: EXO1#-1: 5\u0026prime;-CCATGTGAGGAAGTATAAA-3\u0026prime;, EXO1#-2: 5\u0026prime;-CCAATCTTCTTAAGGGAAA-3\u0026prime;. For siRNA transfection, the cells were plated in 6-well plates overnight and then transfected with siRNA targeting EXO1 using RFectPM transfection reagent (Changzhoubaidai, China). The adenovirus-shKu70 (ADV1, U6/CMV-GFP: 5\u0026prime;-CCAGTGTATCCAAAGTGTGTA-3\u0026prime;) was purchased from GenePharma (Shanghai, China). Cells were plated in 6-well plates overnight and then transducted with adenovirus-shKu70 for one to seven days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. DSB reporter assays\u003c/h2\u003e \u003cp\u003eThe plasmids pimEJ5GFP (EJ5), EJ2GFP-puro (EJ2), and pCBASceI were purchased from Addgene (Cambridge, MA, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.addgene.org/\u003c/span\u003e\u003cspan address=\"https://www.addgene.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A549 cell lines, each with an integrated reporter (EJ2 or EJ5), were generated according to a previously published integration protocol \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. First, siRNA transfections were utilized in the DSB reporter assays: 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e A549-EJ2/EJ5 cells were seeded in a 24-well plate with 30 pmol of each siRNA mixed with 2 \u0026micro;l RfectPM transfection reagent (Changzhoubaidai, China) in 0.5 ml of medium without antibiotic. The cells were cultured for 24 h. Second, cells were transfected with 2 \u0026micro;l Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) mixed with 1 \u0026micro;g of I-SceI endonuclease expression vector (pCBASceI) in 0.5 ml antibiotic-free media. The cells were then cultured for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Survival experiments\u003c/h2\u003e \u003cp\u003eFor the survival experiments, A549, EXO1-depleted A549, A549/DDP, and EXO1-depleted A549/DDP cells (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e) were individually seeded in 96-well plates. Various concentrations of DDP and\u003cem\u003e/\u003c/em\u003eor DNA-PK inhibitor (NU7026) were added to the cells for 24 h. Cell viability was assayed using Cell Counting Kit-8 (CCK-8) (Solarbio, China) and a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Single-cell gel electrophoresis (SCGE) assay\u003c/h2\u003e \u003cp\u003eCells were plated in 12-well plates at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well. The cells were treated with 4 mg\u003cem\u003e/\u003c/em\u003el DDP for 6 h, 10 \u0026micro;M NU7026 for 24 h, or 10 \u0026micro;M NU7026 for 24 h followed by 4 mg\u003cem\u003e/\u003c/em\u003el DDP for 6 h and then analyzed by SCGE at different recovery times, according to published procedures \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Image analysis of at least 50 cells was performed using CometScore software. Data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;50).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Western blot\u003c/h2\u003e \u003cp\u003eCell line extracts were prepared by RIPA lysis buffer (Beyotime, China). SDS loading buffer was added to cell extracts. 30 \u0026micro;g of cell extracts were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with indicated antibodies and detected with chemiluminescent immunoassay (baoguang, China). The following antibodies were used: anti-RBMX (ab190352, Abcam; Cambridge, United Kingdom), anti-EXO1 (16253-1-AP, proteintech; Rosemont, USA), anti-DNA-PKcs (ab133516, Abcam; Cambridge, United Kingdom), anti-Ku70 (ab92450, Abcam; Cambridge, United Kingdom), anti-Ku80 (ab80592, Abcam; Cambridge, United Kingdom), anti-Lig4 (ab193353, Abcam; Cambridge, United Kingdom), anti-Lig1 (ab177946, Abcam; Cambridge, United Kingdom), anti-Lig3 (26893-1-AP, proteintech; Rosemont, USA), anti-PARP-1 (#9532, CST; Danvers, MA, USA), anti-caspase-3 (#9662, CST; Danvers, MA, USA), anti-PCNA(GTX100539, GeneTex; USA), anti-FEN1(GTX101777,GeneTex; USA)anti-ACTIN (66009-1-Ig, proteintech; Rosemont, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Co-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eCells were washed with ice-cold PBS and then lysed in IP buffer (Beyotime, China) supplemented protease inhibitors for 30 min on ice. Cell lysates were removed by centrifugation and the supernatants were incubated with EXO1 antibody magnetic beads (MedChemExpress) overnight at 4\u0026deg;C. Magnetic beads were washed four times with 1 ml of PBST (1\u0026times;PBS\u0026thinsp;+\u0026thinsp;0.5% Triton X-100, pH 7.4). Bound proteins were eluted with 20\u0026ndash;30 \u0026micro;L 2 \u0026times; SDS-PAGE Loading Buffer and subjected to western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Immunofluorescence\u003c/h2\u003e \u003cp\u003eAfter fixation with 4% paraformaldehyde, the cells were permeabilized with PBS containing 0.1% Triton X-100. The cells were stained with 53BP1-, DNA-PKcs-, Ku70-, Ku80-, Lig4-, Lig1-, Lig3-, or PARP-1-specific antibody, washed twice, and then stained with goat anti-rabbit antibody F(ab\u0026acute;)2 fragment conjugated with Alexa Fluor 488 (Carlsbad, California, USA). Nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI; Carlsbad, California, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. In vivo studies\u003c/h2\u003e \u003cp\u003e Animal experiments were approved by the Ethics Committee of Chongqing Medical University. Twenty, 5-week-old male Nu/Nu mice were obtained from Beijing Hua Yikang Biotechnology Co. Ltd. The mice were randomly assigned to four groups (five mice per group). Cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) in 0.2 mL PBS were injected subcutaneously in the groin of each mouse. The volume of the tumor was measured every three days and recorded. DDP and EXO1 inhibitor (EXO1-i) were administered intraperitoneally daily for 3 weeks (Groups: PBS, 3 mg/kg DDP, 10 mg/kg EXO1-i, and 3 mg/kg DDP plus 10 mg/kg EXO1-i) when each tumor reached 0.5 cm in diameter. Tumor volumes were calculated as follows: volume\u0026thinsp;=\u0026thinsp;length \u0026times; width \u0026times; width \u0026times; 0.5. After 4 weeks, all mice were euthanized, and the subcutaneous tumors were weighed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Statistical analysis\u003c/h2\u003e \u003cp\u003eDifferences between the indicated experimental groups were evaluated by one-way ANOVA using SPSS 11.5 software. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Screening of candidate proteins involved in Alt-EJ pathway in vitro\u003c/h2\u003e \u003cp\u003eOur recent study showed that ssO can hijack Ku protein and result in stimulating error-prone Alt-EJ repair of DSBs, since Ku is the core protein to initiate C-NHEJ and to recruit the following repair proteins \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Based on this instant \u003cem\u003ein vitro\u003c/em\u003e Alt-EJ pathway model, to acquire the damage repair related protein pool in Alt-EJ process, we performed a pull-down experiment to harvest proteins recruited to DNA ends for further identification. One end of the dsDNA probe is biotinylated and bound to streptavidin-coated magnetic beads \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. After the introduction of ssO (competitor DNA), Ku protein was bound to the ssO and dislocated from dsDNA \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, the proteins bound at dsDNA ends contains DSBs are harvested and identified.\u003c/p\u003e \u003cp\u003eIn the absence of ssO, we found that Lig4 and DNA-PK (DNA-PKcs and Ku) were specifically bound to the dsDNA probes, clearly separated from the soluble proteins. When most Ku was attached to ssO in soluble extract, PARP-1 was found to bind to the dsDNA probes, demonstrating the competition between the Ku and PARP-1 in binding to dsDNA ends, which is consistence with previous literatures \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, PARP-1 was also recovered on dsDNA probes in the absence of ssO, probably due to excess, non-limiting dsDNA in the assay. Under our conditions, Lig1 involved in the Alt-EJ pathway was not recruited to dsDNA beads (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the molecular weight of the band marked by the red arrow in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB is the same as that of DNA-PKcs, Ku80, and Ku70, respectively. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB are similar to those in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese biochemical results have shown different binding affinity of main known C-NHEJ and Alt-EJ proteins in the presence or absence of ssO. The binding of Ku with ssO prevents the binding of DNA-PK and Lig4-dependent protein complexes with the dsDNA ends, which allows PARP-1 recruitment at dsDNA ends in lung cancer cell extract.\u003c/p\u003e \u003cp\u003eBased on this system, to explore unidentified repair proteins involved in the Alt-EJ pathway, we utilized mass spectrometry to screen the proteins pool recovered on the dsDNA probe in the presence of ssO. Using the bioinformatics Gene Ontology (GO) enrichment method to analyze mass spectrometry data, we obtained 8 enrichments containing 21 genes related to DNA damage repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To identify highly expressed genes in lung cancer cells, the expression of 21 genes in HBE, H520, A549, and A549/DDP cells was quantified by RT-qPCR and further compared with western blot. The results of RT-qPCR showed that RNA binding motif protein X-linked (RBMX) and EXO1 were expressed higher in A549 and A549/DDP cells than that in normal bronchial epithelium HBE cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In this study, Ribosomal protein S27 (RPS27), far upstream element-bingding protein 1 (FUBP1) and transcription elongation factor S-II (TCEA1) were also expressed higher in A549, A549/DDP, or H520 cells. Studies have shown that RPS27, FUBP1, and TCEA1 proteins are involved in DNA damage repair \u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, however, there is no clear research showing that these proteins take participate in DSB repair. POLQ and FEN1 have been identified to be involved in Alt-EJ repair \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Nimonkar et al. showed evidence that EXO1 may participate in the initial end resection step of microhomology-mediated end joining (MMEJ) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Since Alt-EJ events are characterized as microhomology, which require end resection and join the ends at microhomology sequences. The activity of exonuclease is needed in this process. To prove EXO1 participating in Alt-EJ pathway, more detailed experiments need to carry out as below. Apart from candidate EXO1, in addition, Adamson et al. indicated that RBMX participates in HR, but in a PARP-1-dependent manner, suggesting that RBMX may also be a candidate protein in the Alt-EJ pathway \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The western blot results showed that the expression of EXO1 were higher than that in normal cells, which is consistent with the RT-qPCR results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI) .According to the RT-qPCR results and reported studies, we selected RBMX and EXO1 for subsequent investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Recruitment of EXO1 to chromatin in response to radiomimetics in Ku70-deficient lung cancer cells\u003c/h2\u003e \u003cp\u003eTo observe the distribution and colocation of C-NHEJ and Alt-EJ pathway proteins in cells, before and after Cali treatment, we performed immunofluorescence technique. We found that 53BP1, DNA-PKcs, Ku70, Ku80, Lig4, Lig1, Lig3, and PARP-1 proteins were localized to the nucleus, where they were uniformly distributed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After treatment with Cali to induce DSBs, repair proteins aggregated around the broken chromatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results suggest that proteins involved in DNA damage repair are recruited to the ends of DSBs of the broken chromatin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further verify the binding affinity of EXO1 and RBMX to chromatin after radiomimetic treatment in lung cancer cells, we performed fractionation method. This protocol adopts continuous detergent extractions (fractions I\u0026ndash;IV, representing cytoplasm to chromatin) which gradually removing loosely bound proteins from the chromatin, allowing visualization of the association of repair proteins at DSB sites in the chromatin. In Cali treated A549 and A549/DDP cells, as expected, most key proteins of C-NHEJ pathway were released in the early extracted I and II, retained in extraction resistant fractions III and IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). A marker of DSBs, γ-H2AX, was detected in insoluble fraction IV. In contract, Alt-EJ proteins PARP-1, Lig-1 and Lig-3 remained mainly in extractable fractions, with or without Cali treatment, displaying their low affinity for chromatin under this condition. With a similar distribution pattern with Alt-EJ proteins, EXO1 was consistent found in fraction I and II. However, nuclear protein RBMX was distributed differently from the known C-NHEJ and Alt-EJ pathway proteins. It possesses notably constant high affinity with chromatin before or after Cali (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWe planned to investigate the distribution of proteins in the IV fractions after Cali treatment in Ku70-deficient and Ku70-normal cells. To further confirm whether the proteins in the Alt-EJ and C-NHEJ pathways differ in their ability to bind to chromatin when DSBs occur, we used adenovirus-shKu70 to knockdown Ku70 expression by transducing A549 cells. We confirmed high infection efficiency by microscopy (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Infected cells were harvested and subjected to western blots for Ku70 expression after five days. Expression of Ku70 proteins was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then observed the known and candidate repair proteins localization with immunofluorescence and distribution in the four fractions in the Ku70-deficient A549 cells treated with or without Cali for 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In immunofluorescence experiment, DNA-PKcs, Ku80, and Lig4 distributed from the nucleus to the cytoplasm after Ku70 knockdown, while Lig1 and Lig3 partially remained in the nucleus in the Cali treated group, suggesting that these proteins undergo redistribution in the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To observe the binding affinity the proteins, fractionation method was performed as before. In the insoluble fractions, γ-H2AX was present. In A549 cells, in the presence of Ku70, Ku and Lig4 were detected in fractions III and IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Figure. 3C) after Cali treatment. PARP-1 was mainly distributed in soluble fractions (I and II), and some in the less soluble fraction III, along with Lig-1 and Lig-3, probably due to the presence of SSBs in DNA in the system. With Ku70 deficiency, PARP-1 shifted to the insoluble fraction IV, indicating their increased chromatin binding affinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These data clearly showed a shift from C-NHEJ to Alt-EJ when Ku70 expression was limited in A549 cells. We verified whether RBMX and EXO1 participate in the chromatin binding process as those known C-NHEJ or Alt-EJ proteins. Notably, the results showed that the distribution of EXO1 was similar to that of the known Alt-EJ pathway proteins. In Ku70 deficiency condition, EXO1 can be detected accumulated in the insoluble fraction IV. RBMX\u0026rsquo;s distribution was different (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Similar results were confirmed in A549/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These data showed EXO1 binding to the DSB ends under Ku70 deficiency condition, preliminarily indicating that EXO1 may be participate in the Alt-EJ pathway, whereas the recruitment patter of RBMX fail to demonstrate its involvement in Alt-EJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3. EXO1 is involved in the mechanism of Alt-EJ\u003c/h2\u003e \u003cp\u003eAfter demonstrating the recruitment of EXO1 to the radiomimetic-induced DSB, to further explore whether it participates in the DSB repair mechanism and contributes to the ligation effect, we used two chromosome substrates that allowed DSBs to be introduced into. The two reporter substrates were EJ5-GFP \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e which indicates the repair of C-NHEJ, and EJ2-GFP \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, indicates that of Alt-EJ (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, S2B). The cells were transfected with pCBASceI to induce DSBs at the I-SceI site of the reporter plasmid. The percentages of GFP\u0026thinsp;+\u0026thinsp;cells reflected the rate of C-NHEJ or Alt-EJ in the cells, respectively. A549 cells were transfected with negative control siRNA, siRNA-1 targeting EXO1, or siRNA-2 targeting EXO1. Western blot confirmed that the expression of EXO1 was lower in cells transfected with siRNA-1 or siRNA-2 compared to cells transfected with the negative control siRNA (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). In parallel, siRNAs targeting EXO1 were transfected into A549-EJ5 cells and A549-EJ2 cells, followed by 24 h incubation to ensure downregulation of EXO1. The flow cytometry results for GFP\u0026thinsp;+\u0026thinsp;cells are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and the percentage of GFP\u0026thinsp;+\u0026thinsp;cells are quantified in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. Compared to the negative control siRNA-transfected cells, the cells depleted of EXO1 with siRNA-1 exhibited a significantly decreased percentage of EJ2-GFP. Importantly, the level of GFP expressed in A549-EJ2 cells depleted of EXO1 by two different siRNAs, which indicates more suppression of EXO1 expression, was significantly lower than that in cells depleted of EXO1 by one siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In these data, we can conclude that the inhibition of EXO1 compromises the repair of DSB with Alt-EJ pathway in cancer cells, indicating EXO1\u0026rsquo;s contribution in Alt-EJ ligation. Notably, we found that the percentage of EJ5-GFP\u0026thinsp;+\u0026thinsp;cells was significantly different between the control and siRNA-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting that depletion of EXO1 may negatively regulate the C-NHEJ pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether inhibiting of C-NHEJ would increase Alt-EJ in the presence or absence of EXO1, we used DNA-PK inhibitor NU7026 to treat cells and inhibit the C-NHEJ ligation of substrate. In cells pre-treated with NU7026, we found an increase in Alt-EJ ligation of substrate in the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Suppression of C-NHEJ leads to activation of Alt-EJ, which was consistence with previous findings. In contrast, in cells pre-treated with NU7026 and transfected with EXO1 siRNA, the ligation quantity of EJ2-GFP substrate was much lower, compared to the NU7026 treated cells without EXO1 siRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The results showed that in DNA-PK suppression condition, inhibition of EXO1 compromise the C-NHEJ independent ligation, demonstrating that when the C-NHEJ pathway is inhibited, the Alt-EJ pathway relies, at least in part, on EXO1 to repair DSB in lung cancer cells. Altogether, our results exhibit EXO1 participation in DSB repair mechanism of Alt-EJ.\u003c/p\u003e \u003cp\u003eResearch showed that proliferating cell nuclear antigen (PCNA) loads onto double-strand breaks and directly interacts with EXO1\u003csup\u003e35\u003c/sup\u003e. FEN1, Alt-EJ pathway protein, strongly interacts with PCNA to promote DNA repair\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. PCNA interacts with FEN1 and Lig1 to complete Okazaki fragment processing and joining\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Therefore, we speculated that EXO1 may participate in Alt-EJ via interacting with proteins in the Alt-EJ pathway. In order to further confirm this conjecture, we performed the Co-IP experiment. Our results showed that EXO1 interacted with PCNA, FEN1, and Lig1, but not with Ku70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results further confirm that EXO1 participates in the Alt-EJ pathway by combining with FEN1 and Lig1 involved in the Alt-EJ pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4. EXO1-depleted cancer cells are more sensitive to DDP\u003c/h2\u003e \u003cp\u003eExpression of EXO1 is associated with the therapeutic effect of drugs that induce DSBs in tumor cells \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. To clarify the relationship between EXO1 and DDP resistance, we used siRNA to knockdown EXO1 in A549/DDP and A549 cells and then treated them with DDP. Cell survival percentage was observed and evaluated. siRNA-mediated depletion of EXO1 led to an overall higher sensitivity of A549 cells to DDP than A549 cells treated with negative control siRNA at each DDP concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar results were observed in A549/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInhibition of DNA-PK with NU7026 to impede C-NHEJ led to increased levels of Alt-EJ in cells treated with negative control siRNA compared to that in A549-EJ2 cells treated with EXO1-targeted siRNA. This suggests that at least a portion of the DSB ends were shunted to be repaired by Alt-EJ in our cells, according to our reporter plasmid system results. Therefore, we investigated whether the depletion of EXO1 sensitizes A549 and A549/DDP cells to NU7026. A549 and A549/DDP cells were transfected with siRNA against EXO1 and then different concentrations of NU7026 were added. Compared to that of mock-depleted cells, NU7026 pretreated cells exhibited a significant decrease in viability in A549/DDP cells depleted of EXO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eNext, we observed the sensitivity to DDP in A549, EXO1-depleted A549, NU7026-pretreated A549, and NU7026-pretreated EXO1-depleted A549 cells. As expected, the highest sensitivity to DDP was observed in NU7026 pretreated EXO1-depleted A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results indicate that DDP-induced DNA damage is repaired by the EXO1-associated pathway when NU7026 inhibits the C-NHEJ pathway. EXO1-depleted cancer cells are more sensitive to DDP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5. DSBs accumulate in EXO1-depleted cells\u003c/h2\u003e \u003cp\u003eGiven that EXO1-depleted cancer cells are more sensitive to DDP, and this sensitivity can be increased by inhibition of C-NHEJ with NU7026 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), we reasoned that the elevated sensitivity might be related with increased levels of DSBs formation at DNA ends. To confirm this, we measured levels of DSBs using an SCGE assay with wild type (WT) and EXO1-depleted A549 and A549/DDP cells treated with DDP.\u003c/p\u003e \u003cp\u003eWe observed that the levels of DSBs continued to be significantly higher in the EXO1-depleted cells than in the WT cells of A549 and A549/DDP, even after recovering for 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Representative images for each recovery time point after DDP treatment of A549 and A549 EXO1-depleted cells are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. We also found that compared to that in A549 and A549/DDP WT cells treated with the DNA-PK inhibitor NU7026 for 24 h, the levels of DSBs continued to be slightly but significantly higher in the EXO1-depleted A549 and A549/DPP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Furthermore, we observed that the EXO1-depleted cells showed significantly greater persistence of DSBs when they were treated with DDP and NU7026, compared with control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). These results demonstrated that inhibition of EXO1 chemosensitizes cancer cells through promotes the formation of unrepaired DSBs after DDP treatment, especially when C-NHEJ was pharmacologically inhibited.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Depletion of EXO1 inhibits DDP-resistant tumor growth in vivo\u003c/h2\u003e \u003cp\u003eBased on the \u003cem\u003ein vitro\u003c/em\u003e EXO1 inhibition, we examined the effect of the combined treatment of DDP and EXO1-i on an established xenograft model \u003cem\u003ein vivo\u003c/em\u003e. DDP and EXO1-i combination treatment remarkably inhibited tumor growth, consistent with the \u003cem\u003ein vitr\u003c/em\u003eo data (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Images of all tumors are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparison of tumor volumes in the groups revealed that DDP and EXO1-i combination treatment substantially reduced tumor volumes, in agreement with the tumor weight results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, expression of apoptotic protein caspase-3 and 53BP1 was greatly increased in the combination therapy group when compared to that in the two other groups, as assessed by immunohistochemistry. All these data are consistent with the \u003cem\u003ein vitro\u003c/em\u003e data and further confirm the synergistic anticancer effect of DDP and EXO1-i \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eYuan et al. was the first to describe the unexpected property of ssO, which activate Alt-EJ by hijacking Ku protein in the C-NHEJ pathway from DSB ends to make access for Alt-EJ proteins to DNA ends subsequently. The mechanism was extensively demonstrated to verify that it was established as an highly efficient Alt-EJ repair pathway system both in vitro and in cells \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In this study, in lung cancer cells, our pull-down experiments suggested that ssO could prevent Ku from binding to DSB ends while PARP-1 protein in the Alt-EJ pathway was recruited, which is consistent with the author\u0026rsquo;s previous work. We prove the mechanism can be widely applied in multiple cell types to achieve an efficient and convenient Alt-EJ repair pathway shift. Based on the mechanism, EXO1 was identified by mass spectrometry among proteins recruited to the magnetic beads in the presence of ssO, along with other known Alt-EJ proteins as FEN1 and POLQ. In our study, EXO1 was selected as a potential player involvement in Ku-independent Alt-EJ pathway in lung cancer cells. In cellular chromatin fractionation assays, we found the mobilization of EXO1 to insoluble fractions, indicating its recruitment to DNA ends under in vitro condition. We also observed that cells treated with Cali showed significantly increased Ku level and accumulated Lig4 in fraction IV, as well as accumulated Lig1, Lig3, and PARP-1 in fraction Ⅲ. With cell extracts, we further showed that Ku depletion in cells induced a shift from C-NHEJ to Alt-EJ. Using a previously published reporter plasmid assay to design to differentiate DSB ligation with C-NHEJ or microhomology based Alt-EJ \u003csup\u003e18\u0026ndash;20\u003c/sup\u003e, we found that, in EXO1-depleted lung cancer cells, DSB repair with Alt-EJ was attenuated whereas C-NHEJ was enhanced. After treatment with a DNA-PK inhibitor, Alt-EJ increased, indicating that the cells were more dependent on Alt-EJ when C-NHEJ was inhibited. Importantly, we found that inhibition of C-NHEJ fail to enhance Alt-EJ in EXO1-depleted cells, indicating that EXO1 is associated with NHEJ independent microhomology dependent pathway in lung cancer cells.\u003c/p\u003e \u003cp\u003eEXO1 contributes to checkpoint progression and to several DNA repair pathways involved in MMR, translesion DNA synthesis, NER, DSBs repair. By matched whole-genome sequencing data for an extensive study of cancer genome alterations, EXO1 is considered a new cancer-driver genes candidate and associated with survival in colorectal adenocarcinoma \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Exo1\u003csup\u003enull/null\u003c/sup\u003e mice show an increase in chromosomal breaks and base substitution, and predominately develop lymphomas \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and cells depleted EXO1 also show chromosomal instability and hypersensitivity to ionizing radiation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. EXO1\u0026rsquo;s hyper-resection can attenuate both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. DNA-resection by EXO1 is probably inhibited by the DNA binders RPA, Ku70/80, and/or CtIP \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In C-NHEJ, the Ku70/80 heterodimer protects the DNA in a complex with DNA-PKcs for DNA end resection \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Therefore, EXO1 has a limited role in this pathway. In contrast, EXO1 likely collaborates in an Alt-EJ pathway with the WRN in trimming the DNA ends \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. PARP-1, a factor involved in DSB Alt-EJ repair, physically interacts with EXO1 and stimulates EXO1 in its 5\u0026rsquo; excision activity in an in vitro condition \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In liver cancer, EXO1 is significantly upregulated in HCC tumor tissues and that high EXO1 plays a carcinogenic role \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Elevated EXO1 mRNA and proteins expression was detected in lung cancer cells in our study. In ovarian cancer, EXO1 contributed to drug resistance \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In this study, we found that depletion of EXO1 in A549 and A549/DDP cells made them sensitive to DNA-PK inhibitor or DDP, which is related to the presence of persistent DSBs in these cells that were evaluated in SCGE analysis. Importantly, our data suggest that the state of EXO1 can influence the response of tumors to DDP.\u003c/p\u003e \u003cp\u003eBased on our \u003cem\u003ein vitro\u003c/em\u003e results, we examined the \u003cem\u003ein vivo\u003c/em\u003e antitumor effect of DDP and EXO1-i treatment alone or in combination using an established xenograft model. Combination therapy with DDP and EXO1-i significantly increased tumor inhibition rates.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, we have demonstrated the role of EXO1 in Alt-EJ and found that the former contribute to DDP resistance. However, in these cases, the detailed mechanism of EXO1 end excision is still poorly understood. Genetic mutations in excision factors are associated with multiple genetic diseases, predisposition to cancer, and premature aging. Our study may also provide a potential target for enhancing the therapeutic efficacy of chemotherapy by the inhibition of EXO1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eYing Yuan conceived the idea of experiments. Yonghong Wang and Yalin Zhu performed the experiments. Hui Zhang interpreted and analyzed the data. Yonghong Wang wrote the draft of manuscript. Wenli Feng,\u0026nbsp;Ying Yuan\u0026nbsp;and\u0026nbsp;Zhenglan Huang\u0026nbsp;critically revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (No.81703095) and the Innovation Support Program for Overseas Students of\u003c/p\u003e\n\u003cp\u003eChongqing (cx2018142).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003e Informed consent was obtained from all subjects involved in the study. The local ethics committees of the\u0026nbsp;Chongqing Medical University\u0026nbsp;approved the use of the biomaterial and data. The consent is available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication:\u0026nbsp;\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFrit P, Barboule N, Yuan Y, Gomez D, Calsou P (2014) Alternative end-joining pathway(s): bricolage at DNA breaks. 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Med Sci monitor: Int Med J experimental Clin Res 26:e918751. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.12659/msm.918751\u003c/span\u003e\u003cspan address=\"10.12659/msm.918751\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alt-EJ, EXO1, Cisplatin, DNA double-strand break, lung cancer","lastPublishedDoi":"10.21203/rs.3.rs-8486386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8486386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlternative end joining (Alt-EJ) is an error-prone repair pathway of DNA double-strand break (DSB) which functions independent of nonhomologous end joining (NHEJ) core factors, especially Ku protein. As a less studied repair pathway, but not less important one, its involving proteins need further investigation. By using single-stranded DNA oligomers (ssO) displacing Ku protein from damaged DNA ends and facilitating Ku independent DSB repair proteins recruited to DNA ends, we identify EXO1 exonuclease is associated with Alt-EJ and may consider as a novel player participating in Alt-EJ pathway in Ku70-deficient lung cancer cells. Depletion of EXO1 greatly increased cellular sensitivity to Cisplatin (DDP) and DNA-dependent protein kinase (DNA-PK) inhibitor NU7026 \u003cem\u003ein vitro\u003c/em\u003e due to the inhibition of the repair of DSBs. EXO1 inhibition also attenuated the growth of DPP-resistant tumors in the \u003cem\u003ein vivo\u003c/em\u003e mouse model through increasing expression of apoptotic protein caspase-3 and 53BP1. Our results revealed EXO1 participation in DSB repair by Alt-EJ. Suppression of EXO1 provides a chemosensitizing therapeutic option for lung cancer patients of DDP resistance.\u003c/p\u003e","manuscriptTitle":"EXO1 is associated with Ku-independent DNA double strand break repair pathway and the inhibition of EXO1 chemosensitizes DDP resistance lung cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 05:25:55","doi":"10.21203/rs.3.rs-8486386/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"64b3b44f-2653-4761-98f0-434a83b0d147","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T11:25:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 05:25:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8486386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8486386","identity":"rs-8486386","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}