PIF1 helicase promotes break‐induced replication in mammalian cells
2021; Springer Nature; Volume: 40; Issue: 8 Linguagem: Inglês
10.15252/embj.2020104509
ISSN1460-2075
AutoresShibo Li, Hailong Wang, Sanaa E. Jehi, Jun Li, Shuo Liu, Zi Wang, Lan N. Truong, Takuya Chiba, Zefeng Wang, Xiaohua Wu,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoArticle20 January 2021Open Access Source DataTransparent process PIF1 helicase promotes break-induced replication in mammalian cells Shibo Li Shibo Li Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USAThese authors contributed equally to this work Search for more papers by this author Hailong Wang Hailong Wang Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USAThese authors contributed equally to this work Search for more papers by this author Sanaa Jehi Sanaa Jehi Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USAThese authors contributed equally to this work Search for more papers by this author Jun Li Jun Li Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Shuo Liu Shuo Liu Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Zi Wang Zi Wang Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Biomedical Gerontology Laboratory, Department of Health Science and Social Welfare, School of Human Sciences, Waseda University, Tokorozawa, Japan Search for more papers by this author Lan Truong Lan Truong Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Takuya Chiba Takuya Chiba Biomedical Gerontology Laboratory, Department of Health Science and Social Welfare, School of Human Sciences, Waseda University, Tokorozawa, Japan Search for more papers by this author Zefeng Wang Zefeng Wang CAS Key Laboratory of Computational Biology, University of Chinese Academy of Sciences, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xiaohua Wu Corresponding Author Xiaohua Wu [email protected] orcid.org/0000-0003-4947-3047 Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Shibo Li Shibo Li Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USAThese authors contributed equally to this work Search for more papers by this author Hailong Wang Hailong Wang Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USAThese authors contributed equally to this work Search for more papers by this author Sanaa Jehi Sanaa Jehi Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USAThese authors contributed equally to this work Search for more papers by this author Jun Li Jun Li Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Shuo Liu Shuo Liu Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Zi Wang Zi Wang Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Biomedical Gerontology Laboratory, Department of Health Science and Social Welfare, School of Human Sciences, Waseda University, Tokorozawa, Japan Search for more papers by this author Lan Truong Lan Truong Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Takuya Chiba Takuya Chiba Biomedical Gerontology Laboratory, Department of Health Science and Social Welfare, School of Human Sciences, Waseda University, Tokorozawa, Japan Search for more papers by this author Zefeng Wang Zefeng Wang CAS Key Laboratory of Computational Biology, University of Chinese Academy of Sciences, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Xiaohua Wu Corresponding Author Xiaohua Wu [email protected] orcid.org/0000-0003-4947-3047 Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Author Information Shibo Li1, Hailong Wang1, Sanaa Jehi1, Jun Li1, Shuo Liu1, Zi Wang1,2, Lan Truong1, Takuya Chiba2, Zefeng Wang3 and Xiaohua Wu *,1 1Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA 2Biomedical Gerontology Laboratory, Department of Health Science and Social Welfare, School of Human Sciences, Waseda University, Tokorozawa, Japan 3CAS Key Laboratory of Computational Biology, University of Chinese Academy of Sciences, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai, China *Corresponding author (lead contact): Tel: +1 858 784 7910, Fax: +1 858 784 7978; E-mail: [email protected] The EMBO Journal (2021)40:e104509https://doi.org/10.15252/embj.2020104509 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Break-induced replication (BIR) is a specialized homologous-recombination pathway for DNA double-strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP-based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1-dependent BIR is used for homology-initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation-prone AT-rich DNA sequences derived from common fragile sites (CFS-ATs) induce BIR upon replication stress and oncogenic stress, and PCNA-dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC-mediated repair of double-ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease-generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS-ATs. The breast cancer-associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes. SYNOPSIS Break-induced replication (BIR) mediates recombination where DNA strand homology is limited to one end of donor sequence. Here, the helicase PIF1 is found to play a critical BIR role in mammalian cells coping with replication stress. An EGFP-based reporter allows monitoring BIR in mammalian cells. Loss of PIF1 or its helicase activity reduces BIR frequency at induced double-strand breaks (DSBs). BIR activation is controlled differently at DSBs generated by endonucleases versus those caused by replication fork breakage. PIF1 protects structure-prone AT-rich sequences derived from common fragile sites, and its loss is syntheticaly lethal in absence of FANCM. Cells expressing the breast cancer-associated PIF1-L319P mutant are defective in BIR. Introduction DNA double-strand breaks (DSBs) are a major cause inducing chromosome rearrangements, a hallmark of cancer cells (Aguilera & Gomez-Gonzalez, 2008; Negrini et al, 2010). Oncogene expression often results in replication stress, leading to fork collapse and DSB formation (Bartkova et al, 2006; Di Micco et al, 2006), which is one driving force behind genome instability. While DSBs can be repaired by different pathways, homologous recombination (HR) is believed to be the most conserved mechanism to repair DSBs (Paques & Haber, 1999; Jasin & Rothstein, 2013). One principal mechanism of HR is gene conversion (GC), which is utilized when both sides of a DSB are homologous to the donor (Appendix Fig S1A, left). In mitotic cells, GC is initiated by 5′–3′ end resection, followed by invading the 3′ single-strand DNA (ssDNA) end into the homologous template to form a displacement loop (D-loop), after which the 3′ end of the invading strand is used as a primer for new DNA synthesis. In mitotic cells, GC occurs mainly by synthesis-dependent strand annealing (SDSA), wherein the other resected end anneals to the newly synthesized strand once displaced from its template. GC can also occur through the double-strand break repair pathway (DSBR, also called the double Holliday junction [dHJ] pathway), in which the other resected end anneals to the displaced strand of D-loop to form dHJ. Resolution of dHJ produces non-crossover (NCO) and crossover (CO) products. GC tracks are usually short, typically < 100 bps in mammalian cells and 50–300 bps in yeast (Sweetser et al, 1994; Taghian & Nickoloff, 1997; Elliott et al, 1998; Nickoloff et al, 1999; Palmer et al, 2003). Break-induced replication (BIR) is another type of HR mechanism that is used when homology is detected at only one DSB end to the donor sequence (Appendix Fig S1A, right) (Llorente et al, 2008; Anand et al, 2013; Malkova & Ira, 2013). Based on the study from yeast, BIR is initiated by strand invasion to form a D-loop and progresses via D-loop migration. The key difference between GC and BIR is that BIR, but not GC, relies on Pol32, a non-essential subunit of Polδ in yeast, and helicase Pif1 (Lydeard et al, 2007; Deem et al, 2011; Donnianni & Symington, 2013; Saini et al, 2013; Wilson et al, 2013; Sakofsky et al, 2014). Once established, BIR often proceeds for a long distance in yeast and can copy hundreds of kilobases of DNA to the end of a chromosome (Davis & Symington, 2004; Malkova et al, 2005). During BIR, replisomes for repair synthesis often disassociate from the templates, resulting in frequent template switching (Smith et al, 2007). Besides single-ended DSBs (seDSBs), BIR is also activated at double-ended DSBs (deDSBs) when only one DSB end contains homology to the donor or when the distance of the homologies in the donor to the two DSB ends is more than 1–2 kb apart (Appendix Fig S1B, bottom, (Jain et al, 2009; Mehta et al, 2017)). A "recombination execution checkpoint" (REC) was proposed by the Haber group to sense the DSB ends and make the choice of GC or BIR: if the homologies to the two DSB ends are close to each other, GC is activated, but if homology can only be detected at one DSB end, BIR is activated (Jain et al, 2009). However, it is still not clear how REC detects the engagement of DSBs to the donor to make the selection of GC or BIR. Consistent with the role of BIR in repairing seDSBs, the Ira group showed in yeast that BIR is involved in repair of replication fork breakage, although it does not appear to be a primary pathway as adjacent converging forks often complete replication at collapsed forks and suppress BIR (Mayle et al, 2015). In mammalian cells, it has been described that oncogene-induced DNA replication, replication stress-induced DNA repair synthesis in mitosis (mitotic DNA synthesis, MiDAS) and alternative lengthening of telomeres (ALT) exhibit BIR characteristics (Costantino et al, 2014; Minocherhomji et al, 2015; Dilley et al, 2016; Roumelioti et al, 2016; Sotiriou et al, 2016). These important discoveries highlighted the significant roles of BIR in DSB repair especially under replication stress. However, it remains unclear how BIR is activated and operated in mammalian cells and what determines the repair pathway selection between BIR and GC. In this study, we established EGFP-based reporters to monitor GC and BIR in mammalian cells. We demonstrated that at DSBs induced by endonucleases, BIR is used for homology-initiated recombination requiring long track DNA synthesis, but not for short track gene conversion (STGC). However, different from BIR in yeast, which can proceed for more than 100 kbs (Davis & Symington, 2004; Malkova et al, 2005), BIR track length at endonuclease-generated DSBs in mammalian cells rarely exceeds 4 kb, and BIR can be completed by either SDSA or end joining. We also found that POLD3-dependent BIR is activated when forks are broken upon encountering DNA nicks or at common fragile site (CFS)-derived AT-rich sequences (CFS-ATs) upon replication and oncogenic stress. Unexpectedly, at broken forks, POLD3-dependent BIR is established even for STGC involving two DSB ends. PCNA and RFC1 are required for BIR activation, and PIF1 recruitment to collapsed/broken forks is dependent on PCNA. We propose that BIR activation mechanism at collapsed/broken forks is different from that at DSBs generated by endonucleases, and does not require sensing DSB ends for activation. Furthermore, we showed that PIF1 and its helicase activity are important for BIR in mammalian cells, and the breast cancer-associated PIF1 mutant L319P is defective in BIR. PIF1 exhibits a synthetic lethal interaction with FANCM that is important for protecting CFS-ATs. Results Establish an EGFP-based reporter to monitor BIR in mammalian cells In yeast, when the distance between the two homologies in the donor template to the two DSB ends in the recipient is 1–2 kb or longer, BIR is preferentially used over GC to repair these DSBs (Appendix Fig S1B, (Jain et al, 2009; Mehta et al, 2017)). We thus established an EGFP-based reporter (EGFP-BIR-5085, Fig 1A) in mammalian cells to analyze homology-initiated recombination that requires long track of DNA synthesis (0.9–3.8 kb). In this reporter, the EGFP open reading frame is split into the N- and C-terminal fragments (EG and FP) which are linked to one of the two intron sequences (intron 1 and 2) derived from insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) (Wang et al, 2004), respectively. The intron sequences are fused with a 1.3 kb Cypridina luciferase fragment (Luc). To avoid single-strand annealing (SSA), the Luc-FP cassette is placed in front of the EG-Luc cassette that contains two inverted I-SceI sites inserted at the end of the Luc sequence. The reporter was introduced into U2OS cells, and clones with a single copy of the reporter stably integrated into the genome were obtained after Southern blot analysis. Figure 1. PIF1 is important for BIR at endonuclease-generated DSBs Schematic drawing of the EGFP-BIR-5085 reporter and the BIR repair product by SDSA (BIR-SDSA) or end joining (BIR-EJ) which results in EGFP expression after splicing. U2OS (EGFP-BIR-5085) reporter cell line was infected by lentiviruses encoding endonuclease I-SceI or empty vector, followed by puromycin selection (2 µg/ml, 2 days) and assayed for the percentage of EGFP-positive cells by FACS analysis 4 days post-infection. BIR track length was determined in the single EGFP-positive clones derived from U2OS (EGFP-BIR-5085) WT and PIF1 KO reporter cell lines after I-SceI (left) or Cas9/sgRNA (right) cleavage by sequencing the PCR products of repair junctions using genomic DNA. Group means are shown and error bars represent ± SD. Dashed lines (3.8 and 0.9 kb) indicate the upper and lower limits of track length that can be scored by this reporter. U2OS (EGFP-BIR-5085) cells expressing shRNAs for RAD51, POLD3, or shRNA vector (Ctrl) were infected by lentiviruses encoding endonuclease I-SceI. The percentage of EGFP-positive cells was assayed by FACS analysis 4 days later (left). Expression of RAD51 and POLD3 is shown by Western blot analysis (right). U2OS (EGFP-BIR-5085) cells carrying Tet-On Cas9/sgRNA-5085 (Appendix Table S1) and expressing indicated shRNA were treated by Nocodozale (0.3 μM) and 40 h later, Doxycycline (Dox, 5 μg/ml) was added. The percentage of EGFP-positive cells was quantified by FACS analysis 48 h after induction (left). Cell cycles before and after Nocodazole treatment were analyzed by FACS following propidium iodide (PI) staining (middle). Expression of RAD51, POLD3, and RAD52 is shown by Western blot analysis (right). U2OS (EGFP-BIR-5085) cells expressing PIF1 shRNA or shRNA vector (Ctrl; left) or expressing PIF1-WT or E307Q mutant with endogenous PIF1 depleted by shRNA (right) were infected by lentiviruses expressing I-SceI. The percentage of EGFP-positive cells was assayed by FACS analysis 4 days post-infection. PIF1 expression level was determined by qPCR (Appendix Fig S3A), and the expression of PIF1-WT or E307Q mutant is shown in Appendix Fig S3C. U2OS (EGFP-BIR-5085) cells and two PIF1 knocked-out (KO) clones derived from the U2OS (EGFP-BIR-5085) cell line and generated by CRISPR KO were assayed for the percentage of EGFP-positive cells by FACS 4 days after I-SceI lentiviral infection. Schematic drawing of the EGFP-STGC-1731 reporter and the repair product generated by STGC is shown (top). iEGFP: internal EGFP. U2OS (EGFP-STGC-1731) cells expressing RAD51, POLD3, PIF1 shRNA, or shRNA vector (Ctrl) were assayed for EGFP-positive repair events by FACS analysis 4 days post-infection of I-SceI lentiviruses (middle). Expression RAD51 and POLD3 is shown by Western blot analysis (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ****P < 0.0001 and n.s. (not significant) P > 0.05. Source data are available online for this figure. Source Data for Figure 1 [embj2020104509-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Upon I-SceI cleavage in the EG-Luc cassette, the repair is initiated by invading the homologous Luc sequence (1.3 kb long homology) to the Luc-FP cassette on its sister chromatid (Fig 1A). The GC track needs to proceed for 3.8 kb to reach the second homologous sequence outside of the reporter (right-side homology, shown in pink) to complete GC. In mammalian cells, GC with short track length is called STGC, but when the GC track length is longer than 1–2 kb, GC is termed as long track gene conversion (LTGC) (Johnson & Jasin, 2000; Puget et al, 2005). Thus, in our reporter, if the invading strand reaches the second homology locating 3.8 kb away, the repair is completed by LTGC via SDSA. As a result of LTGC, the recipient chromatid would contain a new fused EG-Luc and Luc-FP cassette, which will produce a functional EGFP after splicing (Fig 1A, repair products, BIR-SDSA [LTGC]). However, if the replicating strand is prematurely disassociated from its template before reaching the second homology 3.8 kb away, the newly synthesized DNA end may be ligated to the other end of the original DSB via end joining. In this scenario, if the invading strand has completed the replication of the intron-FP fragment (0.9 kb), green cells can also be produced (Fig 1A, repair products, BIR-EJ [end joining]). As expected, I-SceI expression induced green cell formation in the U2OS (EGFP-BIR-5085) reporter cell line (Fig 1B). We performed PCR and sequencing analysis of the resulted single green clones. Surprisingly, only 1 out of 30 green clones (3.3%) completed replication of 3.8 kb and finished LTGC by using the second end homology (right-side homology, Fig 1A and Appendix Fig S14). For the rest 29 clones (96.7%), replication is aborted before reaching the second end homology, and the disassociated replicating strand is ligated to the second DSB end by end joining. The replication track length of each event is determined (Fig 1C, left), and Southern blot analysis was performed to verify the observation (Appendix Fig S2A). Characterization of the end joining junctions revealed that more than 60% of the end joining events contain 1–5 bp microhomology and more than 10% of the events have 1–8 bp insertions at the repair breakpoints (Appendix Fig S2B), suggesting that microhomology-mediated end joining (MMEJ) is involved in ligating the broken ends when BIR is aborted. We further showed that recombination scored by the EGFP-BIR-5085 reporter is dependent on RAD51 and POLD3 (Fig 1D), consistent with a requirement for RAD51-dependent strand invasion and BIR-specific DNA synthesize of 0.9 to 3.8 kb or longer. Short BIR track length and using end joining to complete BIR in mammalian cells were also observed by the Halazonetis group (Costantino et al, 2014). These studies suggest that BIR replication in mammalian cells is not very processive and usually cannot exceed 4 kb, which is quite different from BIR replication in yeast which can proceed for hundreds of kbs (Davis & Symington, 2004; Malkova et al, 2005). In order to distinguish the two mechanisms for completing BIR in mammalian cells, we term the BIR events that are finished by strand annealing to the second homology end as BIR/SDSA and by end joining as BIR/EJ (Fig 1A). We also showed that like RAD51, inactivation of CtIP, BRCA1, or BRCA2 significantly reduces BIR efficiency scored by the EGFP-BIR-5085 reporter (Appendix Fig S2C, Cas9), consistent with the requirement for end resection and homology-dependent strand invasion in BIR. However, MiDAS, which has been shown to use BIR, is RAD51 independent but requires RAD52 (Bhowmick et al, 2016). We thus examined BIR in mitotic cells using the EGFP-BIR-5085 reporter. Interestingly, BIR in mitotic arrested cells, using 1.3 kb homology for strand invasion, is independent of RAD51 but dependent on RAD52 (Fig 1E), and this is likely due to a suppression of RAD51-mediated HR in mitotic cells (Esashi et al, 2005; Ayoub et al, 2009) (see Discussion). PIF1 is important for BIR in mammalian cells PIF1 is important for promoting BIR in yeast (Wilson et al, 2013). To examine whether PIF1 in mammalian cells is also important for BIR, we silenced human PIF1 by shRNAs in U2OS (EGFP-BIR-5085) cells and showed that depleting PIF1 significantly reduces BIR frequency (Fig 1F, left and Appendix Fig S3A, left), suggesting that PIF1 is also important for BIR in mammalian cells. We also generated PIF1-KO U2OS (EGFP-BIR-5085) cells by CRISPR/Cas9 and showed that BIR frequency is reduced in PIF1-KO cells (Fig 1G and Appendix Fig S3B). In contrast, STGC, as scored using the EGFP-STGC-1731 reporter, which contains ~ 0.3 kb homology on either side of the I-SceI cleavage site, is not reduced after I-SceI cut when PIF1 or POLD3 is depleted by shRNAs (Fig 1H). To determine what are the remaining BIR events in PIF1-KO cells, we analyzed single green clones derived from U2OS (EGFP-BIR-5085) PIF1-KO cells after I-SceI or Cas9 cleavage (Fig 1C). The track length of the remaining BIR events in PIF1-KO cells is significantly reduced with the average track length of 1.5 kb (I-SceI) or 1.4 kb (Cas9) vs 2.2 kb (I-SceI) or 1.9 kb (Cas9) in wild-type (WT) cells. Collectively, these data suggest that PIF1 has a conserved function in BIR to promote long track DNA synthesis in mammalian cells. To examine whether the helicase activity of PIF1 is needed for BIR, we expressed PIF1-WT and the helicase mutant PIF1-E307Q in U2OS (EGFP-BIR-5085) cells and inactivated endogenous PIF1 by shRNAs. We showed that BIR is significantly reduced in the PIF1-E307Q mutant, indicating that the helicase activity of PIF1 is required for its function in BIR (Fig 1F, right and Appendix Fig S3C). PIF1 and RAD51 are required for BIR at Flex1, an AT-rich and structure-prone sequence from CFS, upon replication and oncogenic stress BIR is implicated in replication restart at seDSBs upon fork collapse. Consistent with a role of PIF1 in BIR, we showed that PIF1 depletion significantly increases cell sensitivity to hydroxyurea (HU) and aphidicolin (APH; Fig 2A). We also showed that PIF1-deficient cells are sensitive to other DNA damaging agents which disturb DNA replication, such that ATR inhibitor AZD6738, topisomerase inhibitor camptothecin (CPT), DNA alkylating agent methyl methanesulfonate (MMS), and PARP inhibitor Olaparib (Appendix Fig S4). Single molecule DNA combing analysis further demonstrated that PIF1-KO cells are defective in replication restart after HU treatment (Fig 2B). Figure 2. PIF1 is required for BIR to repair DSBs at broken forks to promote replication restart U2OS WT or PIF1-KO cells were treated with the indicated concentrations of HU (left) or APH (right) for 72 h, and the cell viability assay was performed. U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 h and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110–130 fibers for each experiment (right). Experiments were repeated four times for each sample. Schematic drawing of the EGFP-BIR-5085 reporter and the repair steps leading to the repair product expressing EGFP after Cas9n/sgRNA-5085 expression. U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Track length of single EGFP-positive clones derived from U2OS (EGFP-BIR-5085) cells after Cas9/sgRNA-5085 (n = 47) or Cas9n/sgRNA-5085 (n = 39) expression was analyzed by sequencing of the PCR products from genomic DNA covering the repair junctions. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Significance of the differences was assayed by two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001. Download figure Download PowerPoint To investigate BIR mechanisms in repair of broken replication forks, we used Cas9 nickase (Cas9n: Cas9-D10A) to generate nicks on DNA (Jinek et al, 2012) in the EGFP-BIR-5085 reporter, and when replication encounters the nicks, forks would break, leading to DSB formation (Fig 2C). Both Cas9- and Cas9n-induced HR scored by EGFP-BIR-5085 reporter is RAD51, POLD3, and PIF1 dependent (Fig 2D), consistent with the usage of BIR mechanism. We also showed that Cas9n-induced BIR depends on CtIP, BRCA1, and BRCA2 (Appendix Fig S2C, Cas9n). Interestingly, however, repair track length is much longer at DNA breaks caused by nicks (Cas9n) on replication forks with 27 out of 39 events (69.3%) reached the second homology ends (3.8 kb track length), whereas when DSBs were generated directly by Cas9 cleavage, only two out of 47 events (4.3%) completed 3.8 kb of DNA synthesis and the rest events used BIR/EJ (Fig 2E and Appendix Fig S14). Upon replication stress, replication forks are often collapsed and broken at the genomic loci containing DNA secondary structures. To test whether BIR would be induced upon fork breakage at DNA secondary structures, we engineered a new BIR reporter, EGFP-Flex1-BIR-5086, by replacing the I-SceI site in the EGFP-BIR-5085 reporter with an AT-rich sequence, Flex1, derived from CFS FRA16D (Fig 3A and Appendix Fig S5). Flex1 forms DNA secondary structures at replication forks which cause replication fork stalling and fork collapse, especially under replication stress (Zhang & Freudenreich, 2007; Wang et al, 2014; Wang et al, 2018). Indeed, green cells are accumulated upon HU and aphidicolin (APH) treatment in the U2OS (EGFP-Flex1-BIR-5086) reporter cell line (Fig 3B and Appendix Fig S2D), and depletion of POLD3, PIF1, or RAD51 reduced HU-induced green cell accumulation (Fig 3C and Appendix Fig S2D). CtIP, BRCA1, and BRCA2 are also needed for BIR-mediated repair of DSBs caused by replication stress upon APH or HU treatment (Appendix Fig S2D). Thus, BIR plays an important role in repairing DSBs when forks are broken at structure-prone DNA sequences. We also showed that depleting MUS81 reduces γH2AX accumulation at Flex1 after HU treatment (Appendix Fig S6A) and causes a reduction of Flex1-induced BIR (Appendix Fig S6B), suggesting that MUS81 is involved in cleaving stalled forks at Flex1 and resulted DSBs are repaired by BIR. We analyzed the BIR track length in the EGFP-Flex1-BIR-5086 reporter after APH treatment and found that all 38 green clones (100%) have completed 3.8 kb of DNA synthesis (Appendix Fig S14). We do not know the exact BIR track length beyond 3.8 kb, as it is the maximal BIR track length that can be determined in this reporter. Nevertheless, this study suggests that DNA synthesis track length during BIR is much longer at broken replication forks than that at DSBs generated directly by endonucleases. Figure 3. PIF1 is required for BIR at Flex1 upon replication and oncogenic stress Schematic drawing of the EGFP-Flex1-BIR-5086 reporter. Flex1: 0.3 kb Flex1(AT)34 derived from CFS FRA16D. U2OS (EGFP-Flex1-BIR-5086) cells were treated with or without 2 mM HU for 24 h, and the percentage of EGFP-positive cells was quantified by FACS analysis 3 days after HU removal. U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 h. The percentage of EGFP-positive cells by HU induction was quantified by FACS analysis 3 days after HU removal. U2OS (EGFP-Flex1-BIR-5086) cells were infected by retrovirus expressing H-RAS-V12-FLAG (RAS) or Cyclin E-HA (Cyc E), and the percentage of EGFP-positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). The expression of RAS or Cyclin E is shown by Western blot analysis (bottom). U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3, and PIF1 or shRNA vector (Ctrl) were infected by retroviruses expressing RAS, and the
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