Genetic Evidence for the Involvement of Mismatch Repair Proteins, PMS2 and MLH3, in a Late Step of Homologous Recombination.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease produces the majority of crossovers during meiotic HR. It remains elusive whether mismatch repair factors promote HR in non meiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3 -/- and PMS2 -/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating MLH3 DN/DN and PMS2 EK/EK cells. MLH3 -/- and MLH3 DN/DN cells showed a very similar phenotype, 2.5 times decrease in the frequency of heteroallelic HR-dependent repair of a restriction-enzyme-induced DSBs. PMS2 -/- and PMS2 EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3 DN/DN and PMS2 EK/EK mutations had an additive effect on the heteroallelic HR. MLH3 DN/DN /PMS2 EK/EK cells showed normal kinetics of g -irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and three times decrease in the number of cisplatin-induced sister chromatid exchange (SCE). The ectopic expression of the Gen1 HJ resolvase partially reversed the defective heteroallelic HR of MLH3 DN/DN /PMS2 EK/EK cells. We propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells. DSBs unrepaired but also can cleave intact sister chromatids (78). The following two mouse experiments suggest the critical role played by HJ resolvases in tumor suppression. SLX4 serves as docking sites for the MSH2, MUS81-EME1, SLX1-SLX4, and XPF-ERCC1 endonucleases (78). SLX4 plays a dominant role in preventing carcinogenesis, as evidenced by the data that the loss of SLX4 decreases a median survival time of mice to ~90 days due to enhanced tumorigenesis (79). The MUS81 null mutation reduces the life expectancy of p53 null deficient mice by about 30% due to an increase in carcinogenesis (80). The critical role of MLH3 and PMS2 in the resolution of HJs emphasizes their strong tumor suppressor activities in addition to their function in MMR (21, 22, 81).

A subclass of the MMR proteins is involved in double-strand break (DSB) repair. Firstly, MutS complexes play a role in the rejection of heteroduplex DNA containing insertion/deletion mismatches when the nucleotide sequences of two partner DNAs are not identical (16,17). Second, MutSα may recognize mismatches within the heteroduplex region of the JMs and avoid recombination, collaborating with RecQ helicases (18). Third, MLH1 can affect nonhomologous end-joining (NHEJ) (19), which repairs 80% of the ionizing-radiation-induced DSB in the G2 phase (20). Forth, a subset of MSH and MLH proteins promote meiotic HR, which function is distinct from their MMR functions. MLH1-MLH3, which has a minor role in MMR, is critical for producing meiotic crossover products in mice and Saccharomyces cerevisiae (S. cerevisiae) (21, 22). MLH1, MLH3, and PMS2 are essential for the progression of meiotic HR in mice (23-28).
The role played by the putative endonuclease activity of PMS2 in the resolution of meiotic HR intermediates has not yet been clarified in the mouse or human but recent studies have unveiled new insights into the molecular mechanisms of MLH1-MLH3 and the role of its endonuclease activity (29, 30). Another unsolved question is whether MLH3 and PMS2 promote HR in mammalian somatic cells. There are two major DSB repair pathways in mammalian cells, HR and nonhomologous endjoining (NHEJ). The two pathways differentially contribute to cellular tolerance to anti-malignant therapies. These pathways contribute to tolerance to radiotherapy with HR functioning in the S to G2 phases and NHEJ functioning in the whole cell cycle (46). HR, but not NHEJ, repairs DSBs induced by camptothecin (Top1 poison) and olaparib (poly[ADP ribose]polymerase poison). NHEJ plays the dominant role in repairing DSBs induced by ICRF-193 (catalytic inhibitor of Top2) (47,48). Thus, the sensitivity profile of DSB-repair mutants to these chemotherapeutic agents help to discriminate which repair pathway is compromised in the mutants.
To investigate the role for MLH3 and PMS2 as nucleases in DSB repair of somatic mammalian cells, we inserted point mutation into the DQHA(X)2E(X)4E motif of the endogenous MLH3 and PMS2 genes of the human TK6 B cell line (49) and generated MLH3 D1223N/D1223N and PMS2 E705K/E705K cells. These mutants exhibited increased sensitivities to camptothecin and olaparib, a few times decrease in the frequency of both SCE and the heteroallelic HR, and delayed resolution of g-ray-induced Rad51 foci, indicating a defect in HR in later steps. Surprisingly, their role seems to be mostly independent of MLH1. We conclude that the MLH3 and PMS2 proteins promote DSB repair by HR presumably by processing JMs in human cells. 5 We disrupted the PMS2 and MLH3 genes in TSCER2 cells (50, 51), a TK6 sub-line for measuring heteroallelic HR, generating PMS2 -/and MLH3 -/cells ( Figures S1 and S2). We also generated MSH2 -/cells ( Figures S3A and B), as MSH2 plays a major role in MMR but is not involved in the resolution of HJs in S. cerevisiae (21). MSH2 -/cells were tolerant to an alkylating agent, temozolomide ( Figure S3C), as expected from a defect in MMR (52). PMS2 -/and MLH3 -/cells were sensitive to camptothecin, γ-irradiation, and olaparib ( Figures 1A and B), whereas MSH2 -/cells were tolerant to these damaging agents ( Figures S3D, E, and F). These data suggest the involvement of PMS2 and MLH3 in HR-dependent DSB repair independently of their functioning in MMR or independently of their interaction with MutSα (MSH2-MSH6) or MutSβ (MSH2-MSH3) heterodimers. We generated MLH1 -/cells ( Figure S4) and verified a defect in MMR by confirming the marked tolerance to temozolomide (53) ( Figure S4F). We found no noticeable sensitivity of MLH1 -/cells to γ-irradiation ( Figure 1A). We disrupted the MUS81 gene in wild-type and PMS2 -/-TK6 clones ( Figure S5). The resulting MUS81 -/cells showed a phenotype very similar to that of PMS2 -/cells. MUS81 -/-/PMS2 -/cells showed higher sensitivity to olaparib than MUS81 -/and PMS2 -/cells ( Figure 1C). These observations support the notion that PMS2 and MUS81 act independently of each other in HR-mediated DSB repair.

MLH3 and PMS2 mutants, but not MSH2 and MLH1 mutants, are sensitive to both camptothecin and olaparib
To investigate the catalytic role of MLH3 and PMS2, we inserted point mutations into the endogenous MLH3 and PMS2 genes at the highly conserved DQHA(X)2E(X)4E metal-binding motif. The replacement of the glutamic acid residue in position 705 by lysine (E705K) in human PMS2 completely inactivates its endonuclease activity (15,54,55). Likewise, the D523N and E529K mutations in S. cerevisiae MLH3, which correspond to the D1223N and E1229K mutations in human MLH3, impair both MMR and the resolution of JMs in meiotic HR in mice and Saccharomyces cerevisiae (21, 22, 56-59). We thus generated PMS2 E705K/E705K cells ( Figure S1), and MLH3 D1223N/D1223N and MLH3 E1229K/E1229K cells ( Figure S6). These mutants are hereafter written as PMS2 EK/EK , MLH3 DN/DN , and MLH3 EK/EK cells. The sensitivity profile of PMS2 EK/EK cells was the same as that of PMS2 -/cells ( Figure 1A). Likewise, MLH3 -/-, MLH3 DN/DN , and MLH3 EK/EK cells showed the same phenotype ( Figure 1B). These observations suggest that PMS2 and MLH3 contribute to HR mediated DSB repair as the endonuclease.

MLH3 mutant cells
To monitor DSB repair selectively during the G2 phase when HR is active, we exposed cells to ionizing-radiation and measured the number of chromosomal aberrations in mitotic chromosome spreads at three hours after ionizing-radiation. Only cells that were g-irradiated at the G2 phase, but not S phase, can enter the M phase within three hours (60). This method allows for evaluating the capability of HR to repair DSBs with several times higher sensitivity than the analysis of the girradiation-sensitivity of asynchronous cell populations ( Figures 1A and B). Indeed, g-irradiation increased the number of chromosomal breaks by 1.0 per MUS81 -/cell and only 0.2 per wild-type cell ( Figure 2B). Remarkably, the total numbers of chromosome aberrations induced by g-rays were around ten times higher in the PMS2 and MLH3 mutant cells in comparison with wild-type cells ( Figure 2B). The total number of mitotic chromosome aberrations was significantly higher in MLH1 -/cells, but not in MSH2 -/cells, in comparison with wild-type cells ( Figure 2B). We conclude no significant contribution of canonical MMR involving MSH2 to DSB repair during the G2 phase.
These data suggest that PMS2 and MLH3 significantly contribute to DSB repair as endonuclease.
We counted the number of chromosome aberrations distinguishing chromatid-type breaks (one of the two sister chromatids is broken), isochromatid-type breaks (two sister chromatids are broken at the same sites) and radial chromosomes (comprise the association of two or more chromatids) ( Figure 2A). Ionizing irradiation of RAD54 -/cells caused a more significant increase in the number of chromatid-type breaks than that of isochromatid-type breaks ( Figure 2B). This observation agrees with the role of RAD54 in promoting strand exchange and JM formation. In contrast, MUS81 -/cells showed marked increases in the numbers of isochromatid-type breaks.
Isochromatid-type breaks result from abnormal processing of JMs between broken and intact sister chromatids, as the persistent presence of JMs interferes with local chromosome condensation of both sister chromatids, leading to microscopically visible breakage of the two chromosomes at the same sites (39, 61, 62). Radial chromosomes may be caused by the abnormal separation of JMs containing two sisters, leading to inverted chromosome fusions. Like MUS81 -/cells, the PMS2 and MLH3 mutants showed significant increases in the numbers of both isochromatid-type breaks and radial chromosomes ( Figure 2B). These data suggest that PMS2 and MLH3 promote HR-dependent DSB repair after formation of JMs as does MUS81.
Surprisingly, the MLH1 -/phenotype is not as severe as expected from the phenotypes of the PMS2 and MLH3 mutants, particularly no significant alteration regarding isochromatid-type breaks. A moderate increase in the number of chromatid-type breaks in MLH1 -/cells suggests that the MLH1-MLH3 and MLH1-PMS2 heterodimers may play a minor role in NHEJ-mediated DSB repair, as suggested previously (19). One possible scenario is that an MLH1-independent alternative mechanism of PMS2 and MLH3 might be present in the process observed in this study. These data suggest that PMS2 and MLH3 promote HR-dependent DSB repair after the formation of JMs, as does MUS81. PMS2 EK/EK /MLH3 DN/DN cells showed high sensitivity to camptothecin and g-rays, higher than MLH3 DN/DN , and slightly higher than PMS2 EK/EK , suggesting a prominent role of PMS2 in these assays Figure 1D). PMS2 EK/EK /MLH3 DN/DN cells also showed a higher sensitivity to olaparib than did PMS2 EK/EK and MLH3 DN/DN cells ( Figure 1D). The number of g-ray-induced chromosomal breaks was more than 50% higher in PMS2 EK/EK /MLH3 DN/DN cells than in PMS2 EK/EK and in MLH3 DN/DN cells ( Figure 2). We, therefore, conclude that PMS2 and MLH3 contribute to HR as the endonuclease independently of each other.

MLH3 DN/DN cells
We monitored DSB repair kinetics by measuring the number of gH2AX foci with time after g-

Resolution of g-ray-induced RAD51 foci is delayed in the PMS2 and MLH3 mutant cells
To evaluate if PMS2 and MLH3 act in the early and late steps of HR, we analyzed the formation of Rad51 foci over time after g-irradiation ( Figure 4). The number of Rad51 foci peaked at two hours after g-irradiation in wild-type TK6 cells (60).  Figure 4B). All mutants were less sensitive than RAD54 -/cells. We, therefore, conclude that the PMS2 and MLH3 endonucleases promote HRdependent DSB repair after the polymerization of Rad51 at DSBs.

MLH3 DN/DN and PMS2 EK/EK cells are deficient in heteroallelic HR
To assess the involvement of PMS2 and MLH3 in the resolution of HJs, we measured the frequency of heteroallelic recombination between the allelic thymidine kinase (TK) genes carrying compound heterozygous mutations (47, 50, 60, 63) ( Figure 5A). One of the two allelic TK genes carries an I-Sce1 site, and a mutation in the exon 5 localizes 108 nucleotides downstream of the I-Sce1 site.
When I-Sce1-induced DSBs are repaired by either the gene conversion (HR) that associates with crossover or long-tract gene conversion, it can restore an intact TK gene. These restoration events are detectable by counting the frequency of drug-resistant colonies (50). The HR frequency was 60% smaller in MUS81 -/cells compared with wild-type cells ( Figure 5B), suggesting that a majority of the heteroallelic recombination events involve the formation of HJs. The PMS2 and MLH3 mutants, including MLH3 DN/DN and PMS2 EK/EK cells, showed 60-70% decreases, and PMS2 EK/EK / MLH3 DN/DN and MUS81 -/-/PMS2 -/cells showed further declines in the frequency of crossover events when compared with wild-type cells ( Figure 5B). These observations suggest that the endonuclease-activity of PMS2 and MLH3 may be involved in the resolution of HJs. This function of PMS2 and MLH3 is also independent of MLH1 and MSH2.
We further assessed the involvement of PMS2 and MLH3 in the resolution of HJs by measuring SCE events, crossover-type HR (62, 64,65). To induce SCE, we treated cells with cisplatin, an interstrand crosslinking agent. The number of cisplatin-induced SCE events was measured by subtracting the number of SCE before cisplatin-treatment from the number of SCE post-treatment ( Figure 5C and 5D). The treatment increased the SCE frequency by 11 events per 100 mitotic wildtype cells ( Figure 5D). The number of induced SCE was 50% smaller in MUS81 -/cells when compared with wild-type cells. MLH3 DN/DN and PMS2 EK/EK cells also showed 50% decreases, and PMS2 EK/EK /MLH3 DN/DN cells showed an 80% decrease in the SCE in comparison with wild-type cells ( Figure 5C and 5D). In summary, PMS2 and MLH3 contribute to crossover formation most likely by promoting the resolution of HJs, as does MUS81, but MLH1 is not involved in this process.

The loss of MLH1 does not impair HR-dependent DSB repair
MLH1 physically interacts with PMS2 and MLH3 as heterodimers and thereby stabilizes the two endonucleases (66). Here to evaluate the role of MLH1 in the mitotic HR we have employed five phenotypic assays, (i) sensitivity to camptothecin, γ-irradiation and olaparib ( Figure 1A), (ii) g-ray- Unexpectedly, as mentioned above, all of these phenotypic assays consistently showed that MLH1 -/cells were proficient in HR-mediated DSB repair. We, therefore, conclude that MLH1 is dispensable for the functioning of human MLH3 and PMS2 in mitotic HR. It represents to our knowledge the first example, where MLH1 is not required for the functioning of MLH3 and PMS2.
Indeed, MLH1 has been shown to be required for the functioning of MLH3 and PMS2 in MMR and in meiotic HR in mice (24, 25).
We speculated that the MLH1-independent function of PMS2 and MLH3 in mitotic HR can be achieved through I) homodimer formation, II) forming heterodimer with unknown partner protein which stabilize the PMS2 and MLH3 proteins, and III) MLH3-PMS2 heterodimer. We could not examine these possibilities due to the lack of specific antibodies and no appropriate method of inserting functional tag sequences into PMS2 and MLH3. We therefore investigated PMS2 and MLH3 homodimer and heterodimer formation through 3D-structure modeling using a standard homology modeling pipeline based on the HHpred and RosettaCM methods (67)(68)(69). Structural analysis of the resulting models supports the potential homodimer and heterodimer formation ( Figure S8A, S8B, and S8C).

GEN1.
We reason that if the PMS2 and MLH3 endonuclease activities promote HR by processing HJs, the mutant phenotype of MLH3 DN/DN and PMS2 EK/EK cells could be suppressed by ectopic expression of one of the resolvases described for HJs. We chose GEN1 as the HJ resolvase (70), and used the GEN1 transgene carrying mutations in its nuclear export signal (NES) and fused with the nuclear localization signal (NLS) (71) ( Figure 6A). We added the FLAG tag to this GEN1 transgene and inserted it into the pMSCV retroviral expression vector, which allows for the bi-cistronic expression of the GFP and GEN1 transgenes (72) ( Figure S7). We produced recombinant retrovirus and infected them into TK6 clones. To confirm the expression of the transgene, we performed western blot analyses using an anti-flag antibody ( Figure S7D). We measured the ionizing-radiation sensitivity and calculated LD50, the dose of g-rays that reduced the survival of cells to 50% relative to non-irradiated cells ( Figure 6B). The expression of the GEN1 transgene reversed the ionizingradiation sensitivity of MUS81 -/cells, but not wild-type or RAD54 -/cells. Thus, the GEN1 transgene is able to selectively normalize the defective processing of JMs during HR mediated DSB repair.
The GEN1 transgene restored the tolerance of MLH3 DN/DN , PMS2 EK/EK , and PMS2 EK/EK /MLH3 DN/DN cells to g-rays at least partially ( Figure 6B). The rescue effect of GEN1 transgene was more efficient in PMS2 EK/EK /MLH3 DN/DN double mutant cells compare to MLH3 DN/DN and PMS2 EK/EK cells. In agreement with this finding, the GEN1 transgene significantly reduced the total number of chromosomal aberrations in these mutants as well as MUS81 -/cells ( Figure 6C). Importantly, the GEN1 transgene expression reduced the number of isochromatid-type breaks to a considerably greater extent than that of chromatid breaks ( Figure 6C). The GEN1 transgene increased the frequency of heteroallelic HR in MUS81 -/cells by 60% but had no effect on that in wild-type or RAD54 -/cells ( Figure 6D), suggesting that a substantial fraction of heteroallelic HR involves HJ formation as HR intermediates. The GEN1 transgene restored heteroallelic HR in MLH3 DN/DN , PMS2 EK/EK , PMS2 EK/EK /MLH3 DN/DN , as well as MUS81 -/cells but not wild-type or RAD54 -/cells ( Figure 6D). In summary, the PMS2 and MLH3 endonuclease activities facilitate the separation of HJs.

Discussion
We demonstrate that human PMS2 and MLH3 promote DSB repair by HR in human somatic cells.
Previous studies failed to uncover their role in the repair of X-ray-induced DSBs, presumably because murine primary cells deficient in PMS2 are slightly resistant to ionizing radiation due to defective MMR of damaged nucleotides (73). Strikingly, the defective HR phenotype of the PMS2 and MLH3 mutants derived from the TK6 cell line was as prominent as that of TK6 cells deficient The present study shows compelling genetic evidence for the requirement of the PMS2 and MLH3 endonuclease activity for the efficient resolution of HJs. MLH3 DN/DN and MLH3 -/cells showed the same phenotype in the defective HR ( Figure 2). Likewise, the phenotype of PMS2 EK/EK cells was very similar to that of PMS2 -/cells ( Figure 2). These data indicate PMS2 and MLH3 promote HR as the endonuclease. In the MLH3 DN/DN and PMS2 EK/EK mutants, the initial kinetics of g-ray-induced RAD51 focus formation was normal, whereas its resolution was significantly delayed (Figure 4).
We, therefore, conclude that the PMS2 and MLH3 endonuclease activities promote a late step of HR, most likely after the formation of JMs. The MUS81 -/-, MLH3 DN/DN , and PMS2 EK/EK mutants all showed a ~40% decrease in the frequency of cisplatin-induced SCEs ( Figure 5D). Furthermore, ectopic expression of GEN1, a typical HJ resolvase, reversed the defective heteroallelic HR of MUS81 -/-, MLH3 DN/DN , and PMS2 EK/EK cells by 30% -50% ( Figure 6). In conclusion, the endonuclease activity of PMS2 and MLH3 process HJs generating both crossover and noncrossover products.
The PMS2-MLH1 and MLH3-MLH1 heterodimers are involved in both MMR and meiotic HR in S. cerevisiae and mice (21, 24, 25, 66). Unexpectedly, we observed that only PMS2 and MLH3, but not MLH1, are involved in HR in human somatic cells. The PMS2 and MLH3 proteins may form homodimers and heterodimers when they are involved in HR in the same manner as the MutL homologs form heterodimer mediated by their C-terminal region (14). Indeed, homodimers of yeast Mlh1 have been reported, and an increase of their formation can inhibit MMR (74). In addition, in support of a possible heterodimer formation, a recent study in budding yeast found coimmunoprecipitation of Mlh3 with Pms1 (the equivalent of PMS2) (ref59 BioRxiv). The crystal structure of the C-terminal region of human MLH1 (pdb code 3RBN) showed that human MLH1 could form homodimers with the same residues involved in the heterodimer formation (14). The HHpred and RosettaCM analysis also suggested that the homodimer as well as heterodimer formation is possible ( Figure S8). Future studies will demonstrate the homodimer and heterodimer formation as well as the interaction with an unidentified partner protein.
MLH3 and PMS2 have strong tumor suppressor activities, and it is believed that these activities are attributable exclusively to their functioning of MMR (75). The current study suggests that these endonucleases may contribute to tumor suppression also by promoting the resolution of HJs. The In this study, we characterized a major role of MLH3 and PMS2 in the DSB repair that is independent of MSH2 and MLH1. These results highlight an additional layer of the multifunctional role played by the MMR proteins (66). Studies on the molecular mechanisms of the process identified here will allow deciphering if this process is mediated by the homodimeric form of PMS2 and MLH3, complex with not yet characterized partners or a new pathway of DSB repair.

Cell clones
All the clones used in this study are summarized in Table 1.

Generation of human PMS2 −/− TK6 B cells
To generate a pair of TALEN expression plasmids against the PMS2 gene, we used a Golden Gate hours later, cells were seeded into 96-well plates with both hygromycin and puromycin antibiotics for two weeks. The gene disruption was confirmed by genomic PCR using primers P1, P2, P3, P4 ( Figure S1B) and RT-qPCR using primers P5 and P6 ( Figure S1C). All primers used in this study are shown in Table S1.

Generation of nuclease dead human PMS2 E705K/E705K TK6 B cells 14
To generate nuclease dead human PMS2 E705K/E705K TK6 B cells, we designed a guide RNA targeting intron sequence upstream of 12 th exon using the Zhang CRISPR tool (85) and gene-targeting constructs. The CRISPR-target site is depicted in Figure S1C. The gene-targeting constructs were generated using SLiCE (Seamless Ligation Cloning Extract). The genomic DNA was amplified with primers F3 and R3 from the PMS2-gene locus and the PCR product was used as template DNA for amplifying the 5' arm. The 5'-arm was amplified using primers F4 and R4, where each primer shared 20-base pair-end homology with the insertion site of the vector. The sequence intended as the 3' arm of the PMS2 targeting construct was amplified by PCR as two fragments using overlapping primers (F5 and R5) and that included a point mutation to change codon 705 from glutamic acid to lysine.
The two fragments were then combined by chimeric PCR to yield the 3' targeting arm including the codon 705 mutation. The 3'-arm was amplified using primers F6 and R6, where each primer shared 20-base pair-end homology with the insertion site of the vector. Both vectors, DT-ApA/neo and DT-ApA/hygro, were linearized with NotI and XbaI. All the fragments of the vectors and inserts were purified using a QIAquick gel extraction kit (QIAGEN, Netherlands). The gene-targeting constructs were generated in a single reaction mixture containing DT-ApA/neo or DT-ApA/hygro vectors, 5'and 3'-arms, and 2×SLiCE buffer (Invitrogen, US) and incubated for 30 min at room temperature. 6 µg of CRISPR and 2 µg of each gene-targeting vector were transfected into 4×10 6 TK6 cells using the Neon Transfection System (Life Technologies, US). After electroporation, cells were released into 20 ml drug-free medium containing 10% horse serum. Forty-eight hours later, cells were seeded into 96-well plates for selection with both neomycin and hygromycin antibiotics for two weeks. The gene disruption was confirmed by RT-PCR using primers F7 and R7 followed by direct sequencing ( Figures S1D and S1E). The drug resistance markers are flanked by loxP sites, and were thus excised from PMS2 E705K/E705K cells by transient expression of cre-recombinase, leading to the generation of PMS2 E705K/E705K cells.

Generation of human MLH3 −/− TK6 B cells
To disrupt the MLH3 gene, we designed a guide RNA targeting the sixth exon using the Zhang CRISPR tool (85) and gene-targeting constructs. The CRISPR-target site is depicted in Figure S2A.
The gene-targeting constructs were generated using SLiCE (Seamless Ligation Cloning Extract).
The genomic DNA was amplified with primers F8 and R8 from the MLH3-gene locus and the PCR product was used as template DNA for amplifying the 5'-and 3'-arms. The 5'-arm was amplified using primers F9 and R9 and the 3'-arm was amplified using primers F10 and R10, where each primer shared 20-base pair-end homology with the insertion site of the vector. Both vectors, DT-and inserts were purified using a QIAquick gel extraction kit (QIAGEN, Netherlands). The genetargeting constructs were generated in a single reaction mixture containing DT-ApA/neo or DT-ApA/hygro vectors, 5'-and 3'-arms, and 2×SLiCE buffer (Invitrogen, US) and incubated for 30 min at room temperature. 6 µg of CRISPR and 2 µg of each gene-targeting vector were transfected into 4×10 6 TK6 cells using the Neon Transfection System (Life Technologies, US). After electroporation, cells were released into 20 ml drug-free medium containing 10% horse serum.
Forty-eight hours later, cells were seeded into 96-well plates for selection with both neomycin and hygromycin antibiotics for two weeks. The gene disruption was confirmed by RT-PCR using primers F11 and R11 ( Figure S2B), and by Southern blot analysis with a 0.6 kb probe amplified by PCR from genomic DNA using F12 and R12 ( Figure S2C). The genomic DNA of the candidate clones was digested with EcoRI for Southern blot analysis.

Generation of human MSH2 −/− TK6 B cells
To disrupt the MSH2 gene, we designed a guide RNA targeting the 4 th exon using the Zhang CRISPR tool (85) and gene-targeting constructs. The CRISPR-target site is depicted in Figure S3A.
The gene-targeting constructs were generated using SLiCE (Seamless Ligation Cloning Extract).
The genomic DNA was amplified with primers F22 and R22 from the MSH2-gene locus and the PCR product was used as template DNA for amplifying the 5'-and 3'-arms. The 5'-arm was amplified using primers F23 and R23 and the 3'-arm was amplified using primers F24 and R24, where each primer shared 20-base pair-end homology with the insertion site of the vector. Both vectors, DT-ApA/neo and DT-ApA/puro, were linearized with AflII and ApaI. All the fragments of the vectors and inserts were purified using a QIAquick gel extraction kit (QIAGEN, Netherlands).
The gene-targeting constructs were generated in a single reaction mixture containing DT-ApA/neo or DT-ApA/puro vectors, 5'-and 3'-arms, and 2×SLiCE buffer (Invitrogen, US) and incubated for 30 min at room temperature. 6 µg of CRISPR and 2 µg of each gene-targeting vector were transfected into 4×10 6 TK6 cells using the Neon Transfection System (Life Technologies, US).
After electroporation, cells were released into 20 ml drug-free medium containing 10% horse serum. Forty-eight hours later, cells were seeded into 96-well plates for selection with both neomycin and puromycin antibiotics for two weeks. The gene disruption was confirmed by genomic PCR using primers F25, F26 and R25 ( Figure S3).

Generation of human MLH1 −/− TK6 B cells
To disrupt the MLH1 gene, we designed a guide RNA targeting the 8 th exon using the Zhang CRISPR tool (85) and gene-targeting constructs. The CRISPR-target site is depicted in Figure S4A.
The gene-targeting constructs were generated using SLiCE (Seamless Ligation Cloning Extract).
The genomic DNA was amplified with primers F27 and R27 from the MLH1-gene locus and the PCR product was used as template DNA for amplifying the 5'-and 3'-arms. The 5'-arm was amplified using primers F28 and R28 and the 3'-arm was amplified using primers F29 and R29, where each primer shared 20-base pair-end homology with the insertion site of the vector. Both vectors, DT-ApA/neo and DT-ApA/puro, were linearized with AflII and ApaI. All the fragments of the vectors and inserts were purified using a QIAquick gel extraction kit (QIAGEN, Netherlands).
The gene-targeting constructs were generated in a single reaction mixture containing DT-ApA/neo or DT-ApA/puroro vectors, 5'-and 3'-arms, and 2×SLiCE buffer (Invitrogen, US) and incubated for 30 min at room temperature. 6 µg of CRISPR and 2 µg of each gene-targeting vector were transfected into 4×10 6 TK6 cells using the Neon Transfection System (Life Technologies, US).
After electroporation, cells were released into 20 ml drug-free medium containing 10% horse serum. Forty-eight hours later, cells were seeded into 96-well plates for selection with both neomycin and puromycin antibiotics for two weeks. The gene disruption was confirmed by Southern blot analysis (genomic DNA was digested with SphI) with a 0.52 kb probe amplified by PCR from genomic DNA using F30 and R30 ( Figure S4B and S4C). The candidate clones were further confirmed by RT-PCR using primers F31 and R31 ( Figure S4D) and Western blot analysis ( Figure S4E).

Generation of human MUS81 −/− TK6 B cells
To generate a pair of TALEN expression plasmids against the MUS81 gene, we used a Golden Gate TALEN kit and a TAL effector kit (Addgene, US) (83,84). The TALEN target sites are shown in Figure S5A. The gene-targeting constructs were generated from the genomic DNA of TK6 cells by amplifying with primers SacI-flanked F19 and BamHI-flanked R19 for the 5'-arm and BamHIflanked F20 and R20 for the 3'-arm. The 3'-arm PCR products were cloned into pCR-Blunt II-TOPO vector. The 5'-arm PCR products were cloned into the SacI site of the pCR-Blunt II-TOPO vector containing 3'-arm. The BamHI fragment containing either the bsr R or puro R gene was cloned into the BamHI site between the 3'-arm and the 5'-arm in the pCR-Blunt II-TOPO vector. 10 µg TALEN-expression plasmids and 10 µg linearized gene-targeting vectors were transfected into 10×10 6 TK6 cells using the Bio-Rad Gene Pulser II Transfection System at 250 V and 950 µF.
After electroporation, cells were released into 20 ml drug-free medium containing 10% horse serum. 48 hours later, cells were seeded into 96-well plates with both blasticidin and puromycin antibiotics for two weeks. The genomic DNAs of the isolated clones resistant to both hygromycin and puromycin were digested with DraI for Southern blot analysis. A 0.6 kb probe was generated by PCR of genomic DNA using primers F21 and R21 ( Figure S5B).

Generation of nuclease dead human MLH3 D1223N/D1223N and MLH3 E1229K/E1229K TK6 B cells
To generate nuclease dead human MLH3 EK/EK and MLH3 DN/DN TK6 B cells, we designed a guide RNA targeting intron sequence upstream of seventh exon using the Zhang CRISPR tool (85) and gene-targeting constructs. The CRISPR-target site is depicted in Figure S6. The gene-targeting constructs were generated using SLiCE (Seamless Ligation Cloning Extract). The genomic DNA was amplified with primers F13 and R13 from the MLH3-gene locus and the PCR product was used as template DNA for amplifying the 5' arm. with NotI and XbaI. All the fragments of the vectors and inserts were purified using a qiaquick gel extraction kit (QIAGEN, Netherlands). The gene-targeting constructs were generated in a single reaction mixture containing DT-ApA/neo or DT-ApA/hygro vectors, 5'-and 3'-arms, and 2×SLiCE buffer (Invitrogen, US) and incubated for 30 min at room temperature. 6 µg of CRISPR and 2 µg of each gene-targeting vector were transfected into 4×10 6 TK6 cells using the Neon Transfection System (Life Technologies, US). After electroporation, cells were released into 20 ml drug-free medium containing 10% horse serum. Forty-eight hours later, cells were seeded into 96well plates for selection with both neomycin and hygromycin antibiotics for two weeks. The site directed mutagenesis was confirmed by genomic PCR using primers F18 and R18 followed by direct sequencing (Figures S6C and D). The drug resistance markers are flanked by loxP sites, and were thus excised from MLH3 DN/DN and MLH3 EK/EK cells by transient expression of crerecombinase, leading to the generation of MLH3 DN/DN and MLH3 EK/EK cells.

Colony-survival assay
To measure sensitivity, cells were treated with camptothecin (Topogen, Inc, US), olaparib (Funakoshi, Japan) and irradiated with ionizing radiation ( 137 Cs). Cell sensitivity to these DNAdamaging agents was evaluated by counting colony formation in methylcellulose plates as described previously (86).

Heteroallelic crossover analysis
The human lymphoblastoid cell line TSCER2 is a TK6 derivative with an I-Sce1 site inserted into the TK locus (50, 51). TSCER2 cells are compound heterozygous (TK -/-) for a point mutation in exons 4 and 5. DSB occurring at the I-Sce1 site results in homologous recombination between the alleles and produces TK-proficient revertants (TK +/-). 4×10 6 TK6 cells were transfected with 6 µg I-Sce1 expression vector using the Neon Transfection System (Life Technologies, US) with 3X pulse at 1350 V and with 10 msec pulse width and released into 20 ml drug-free medium containing 10% horse serum. After 48 hours, cells were seeded as 1x10 6 cells per 96-well plates, with 2'deoxycytidine (Sigma D0776), hypoxanthine (Sigma H9377), aminopterin (Sigma A3411) and thymidine (Sigma T9250) (CHAT for TK-revertants) medium. Drug resistant colonies were counted 2 weeks later.

Chromosomal aberrations analysis
TK6 cells were irradiated with 1 Gy of IR. The cells were then treated with 0.1 µg/ml colcemid (GIBCO-BRL, Grand Island, NY) and incubated at 37 O C for 3 hours. Experimental conditions for chromosomal aberration analysis were as described previously (72). Briefly, harvested cells were treated with 1 ml of 75 mM KCl for 15 min at room temperature and fixed in 5 ml of a freshly prepared 3:1 mixture of methanol/acetic acid. The cell suspension was dropped onto a glass slide and air-dried. The slides were stained with 5% Giemsa solution (NacalaiTesque) for 10 min and air-dried after being rinsed carefully with water. All chromosomes in each mitotic cell were scored at 1,000× magnifications. A total of 50 mitotic cells were scored for each group using a microscope.

Sister chromatid exchange (SCE) analysis
TK6 cells were incubated with or without cisplatin (2 µM). After one hour, cells were washed and released into BrdU (100 mM) containing media. Cells were incubated for 2 more cell cycle and treated with Colcemid (0.1 mg/ml) for 3 hours before being harvested. Metaphase chromosomes were prepared and assayed for SCEs as previously described (64).

Construction of Flag-tagged hGen1 with nuclear localization signal expressing TK6 cell lines
Flag-tagged hGen1-NES (4A)-NLS + expressing TK6 cells were generated using a genetically modified retroviral vector as described ( Figure S7) (72). Briefly, the coding sequence for hGen1-NES (4A)-3xNLS + -3xFlag was cloned into the pMSCV retroviral expression vector (Clontech) ( Figure S7A). The newly engineered retroviral expression vector was co-transfected into human 293T cells with a helper plasmid (pClampho) expressing the viral gag, pol and env proteins to produce viral supernatant. The viral supernatant was collected after 48h and used to transduce into wild-type, PMS2 EK/EK , MLH3 DN/DN , PMS2 EK/EK /MLH3 DN/DN , MUS81 -/-, and RAD54 -/-TK6 mutant strains ( Figure S7B). The efficiency of each step was assessed by quantifying the number of cells expressing GFP ( Figure S7C). The expression of hGen1-NES (4A)-3xNLS + -3xFlag was further confirmed by western blot (WB). Experimental conditions for WB analysis were as described previously (87). The following antibodies were used: anti-Flag antibody for overnight at 4°C and anti-mouse IgG HRP-linked antibody for 1h at room temperature was used as the primary and secondary antibodies respectively ( Figure S7D).

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The subtracted numbers of breaks before the exposure from breaks after the exposure (Lower panel). Error bars represent propagation of error.