rescues ssDNA gaps PNAS PLUS generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

Wenjian Ma, James W. Westmoreland, and Michael A. Resnick1

Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved June 21, 2013 (received for review January 26, 2013) Repair of DNA bulky lesions often involves multiple repair path- As we and others have reported, DSBs can be formed as ways such as nucleotide-excision repair, translesion DNA synthesis secondary products during processing of ssDNA lesions arising (TLS), and homologous recombination (HR). Although there is con- from agents such as methyl methanesulfonate (MMS) (8-10) at siderable information about individual pathways, little is known doses that result in closely opposed lesions. Because NER can about the complex interactions or extent to which damage in produce ssDNA gaps of ∼30 nt for a variety of bulky lesions, single strands, such as the damage generated by UV, can result in there is a greater likelihood of secondary generation of DSBs double-strand breaks (DSBs) and/or generate HR. We investigated than with base-excision repair, which generates short resection the consequences of UV-induced lesions in nonreplicating G2 cells regions. However, gap formation and subsequent refilling during of budding yeast. In contrast to WT cells, there was a dramatic NER are tightly coordinated, with repair synthesis starting after increase in ssDNA gaps for cells deficient in the TLS polymerases η incision on the 5′ side of the lesion (which precedes the 3′ in- (Rad30) and ζ (Rev3). Surprisingly, repair in TLS-deficient G2 cells cision), resulting in rapid completion of repair (11, 12). This required HR repair genes RAD51 and RAD52, directly revealing a redundancy of TLS and HR functions in repair of ssDNAs. Using coordination might prevent the conversion of ssDNA gaps into a physical assay that detects recombination between circular sister more deleterious DSBs, thereby preserving genome stability. In GENETICS chromatids within a few hours after UV, we show an approximate the event that long gaps are produced, surveillance mechanisms three-fold increase in recombinants in the TLS mutants over that in including checkpoint activation (13, 14) may prevent cell-cycle WT cells. The recombination, which required RAD51 and RAD52, progression until repair has been completed. does not appear to be caused by DSBs, because a dose of ionizing The repair of bulky lesions by NER poses another risk when radiation producing 20 times more DSBs was much less efficient lesions are closely opposed and the repair polymerase encoun- than UV in producing recombinants. Thus, in addition to revealing ters a lesion on the template strand preventing further synthesis. TLS and HR functional redundancy, we establish that UV-induced In the case of DNA replication, lesions on template strands are recombination in TLS mutants is not attributable to DSBs. These tolerated by translesion DNA synthesis (TLS) polymerases that findings suggest that ssDNA that might originate during the repair provide error-prone or error-free bypass of the blocking lesions of closely opposed lesions or of ssDNA-containing lesions or from (15, 16). TLS enables NER to process a variety of bulky lesions, uncoupled replication may drive recombination directly in various including drug-induced DNA crosslinks (5, 17). With increases in species, including humans. dose, TLS could take on an even more important role in repair, because there would be increased likelihood of lesions in the NA bulky lesions, such as UV-induced pyrimidine dimers template strand. In addition, the need for TLS increases with gap Dand interstrand crosslinks caused by some cancer drugs, can size, because there is increased likelihood of encountering a be recognized and processed by nucleotide excision repair lesion during repair synthesis. (NER). Because of their important links with human patholo- gies, including cancer, the underlying repair mechanisms and Significance their impact on genome instability have been studied extensively (1, 2). The repair of bulky lesions is initiated by NER, a versatile DNA damage challenges genome integrity. Bulky DNA lesions, repair system comprising up to ∼30 proteins/enzymes that are which are subject to nucleotide-excision repair, induce homol- well conserved from yeast to mammals (3). The major steps in ogous recombination (HR). However, because there is no direct NER include damage recognition, followed by endonucleolytic generation of double-strand breaks (DSBs), the underlying incisions at the 5′ and 3′ sides of the lesion to remove a 25- to 30-nt mechanism has been obscure. By investigating UV-induced oligonucleotide containing the damage, and DNA synthesis and lesions in nonreplicating G2 cells of budding yeast, we found ligation to fill the gap, thereby returning the DNA region to its that translesion DNA synthesis (TLS) and HR are redundant in original state (3, 4). repair. Using a physical assay that detects recombination be- Repair of bulky lesions often is associated with homologous tween circular sister chromatids, we establish that UV-induced recombination (HR), as suggested for the removal of DNA in- recombination is not attributable to DSBs but instead is asso- terstrand crosslinks (5), likely accounting for subsequent genome ciated directly with expanded ssDNA gaps and is increased in instability. Because there is no direct generation of double-strand cells defective in TLS. breaks (DSBs), the underlying mechanism of recombination, Author contributions: W.M. and M.A.R. designed research; W.M. performed research; including initiation, is generally not well understood. Single- W.M. and J.W.W. contributed new reagents/analytic tools; W.M. and M.A.R. analyzed strand nicks and ssDNA regions arising during NER near rep- data; and W.M. and M.A.R. wrote the paper. lication forks could result in replication fork collapse in S-phase The authors declare no conflict of interest. and lead to recombination (6, 7). However, there is relatively This article is a PNAS Direct Submission. little information about the direct induction of recombination 1To whom correspondence should be addressed. E-mail: [email protected]. by bulky lesions during nonreplicating G1 or G2 stages of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cell cycle. 1073/pnas.1301676110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1301676110 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 We have addressed the role that recombination might play in absence of either or both TLS polymerases in NER-proficient the repair of lesions produced by UV (UV-C) in nongrowing G2 cells has little effect on the sensitivity of G2 cells, indicating that cells, especially under conditions of reduced TLS, when large a large amount of damage (∼15,000 CPDs) (25) is well tolerated gaps might be expected. Using physical assays that detect gaps as in the absence of TLS. Also, there is high UV tolerance in G2 well as recombinational linkage between sister chromatids that cells that are HR deficient because of a lack of either Rad52 or are circular (18), we establish that HR and TLS have redundant Rad51. However, the double TLS/recombination mutants that roles in maintaining chromosome stability after UV irradiation, lack the TLS polymerase Rad30 and either Rad51 or Rad52 are likely because of the ability to repair ssDNA gaps containing ∼10-fold more UV sensitive. Loss of an additional TLS poly- a lesion. Unexpectedly, DSBs appear to have a minor role, at merase Rev3 (i.e., rev3Δ rad30Δ rad52Δ) increases the sensitivity most, in the molecular changes. by another ∼100-fold, demonstrating that the TLS and HR sys- tems have an important role in preventing UV-induced killing of Results G2 cells even though NER is functional. These results suggest Lack of TLS and Recombinational Repair Greatly Increase UV Sensitivity that TLS and HR are redundant in protecting G2 cells from UV of G2 Cells. Two types of DNA lesions are generated by UV irradiation. As described in the following sections, we in- irradiation, cyclobutane pyrimidine dimers (CPDs) and 6-4 vestigated the repair processes in the G2-arrested cells and pyrimidine-pyrimidone products (6-4PPs) (19, 20). They are found that the repair occurred within a few hours after UV ir- subject to NER and block replication if unrepaired. In yeast, radiation. Because UV-irradiated cells that are resuspended in η η medium without nocodazole remain in G2 for at least 2 h (see as well as in most , the TLS polymerases (Pol ) + ζ ζ legend of Table S1), the resistance of cells that are at least TLS and (Pol ) provide replication bypass, allowing duplication + of the genome. The Pol η of the budding yeast Saccharomyces or HR in Fig. 1 is attributed to events in G2 (i.e., before cell- cerevisiae, which is encoded by the RAD30 gene, can bypass CPDs cycle progression). accurately (21). The TLS error-prone Pol ζ, a two-subunit enzyme TLS Deficiency Leads to Accumulation of ssDNA Gaps and Reduced encoded by REV3 (catalytic) and REV7 (accessory), can bypass Chromosome Repair After UV Irradiation. To address the roles that a broad range of lesions including 6-4PPs (22). TLS and HR play in the toleration of UV-induced damage in Here, we examine the response to UV irradiation of haploid fi nocodazole-arrested G2 cells, we followed whole-chromosome budding yeast mutants de cient in TLS and recombination re- changes using pulsed-field gel electrophoresis (PFGE), which pair. All yeast mutants used were derived from haploid strains can display the yeast karyotype that extends from ∼200 kb to containing a circular form of chromosome III (Chr III) (23). We ∼2,200 kb. Beginning shortly after exposure to 50 J/m2, there fi 2 rst determined survival after UV-C irradiation (50 J/m ) of cells were reductions in the intensity of chromosome bands that were that were nocodazole-arrested in the G2 stage of the cell cycle size dependent—the bigger the chromosome, the greater the > ( 90% arrest; see Table S1). Previously, McDonald et al. (24) reduction (Fig. 2A). During the subsequent incubation of cells in showed that the combination of TLS/recombination rich medium (YPDA) containing nocodazole, the intensities greatly increased the UV sensitivity of stationary-phase cells decreased even further over the first hour. This trend reversed (which were likely to be mostly in G1). As shown in Fig. 1, the over the next hours with nearly complete restitution by 4 h after irradiation. Interestingly, although there was a decrease in chromosome bands, there was no apparent fragmentation based on a lack of “smearing” below each band, which would be expected with random DSB breaks resulting from ionizing radi- ation (Fig. 2C). Instead, slow-moving DNA (SMD) appeared just below the well but disappeared almost completely by 4 h after UV irradiation. The SMD had been observed earlier by Gian- nattasio et al. (14) in yeast cells that were UV-irradiated in G1, but in their study there was no reduction in the SMD over the course of the study and nor was there any restitution of chro- mosomes (comparable results were obtained with G1 cells of the strains used in the present study). We also observed SMD during the repair of MMS lesions in apurinic/apyrimidinic AP endo- nuclease-deficient yeast G2 cells (9). SMD was proposed to re- sult from the formation of repair intermediates containing long ssDNA regions as previously was established for G1 cells (14). The amount of SMD was increased in cells lacking RAD30 (Fig. 2A) as well as in cells lacking the TLS Pol ζ (Rev3; Fig. S1). We note that SMD can vary depending on the amount that is retained in the well. However, the differences between WT cells and TLS mutants become clearer when examining the relative loss of chromosomes, and the difference is especially evident for the larger chromosomes (Fig. 2A and Fig. S1). Because there was little, if any, loss of the smallest chromosomes, Chr I and Chr VI, it was possible to quantitate the relative amount of SMD across experiments by determining the ratio of SMD/(Chr I+VI). There was a consistent and statistical difference between WT cells and TLS mutants (Fig. 2D). The chromosome bands were restituted gradually with time but with differing efficiency, more slowly in ad30Δ rev3Δ rad30Δ rev3Δ Fig. 1. Cell survival after UV irradiation in G2. Logarithmically growing cells r and mutants than in WT cells. In were arrested in G2 with nocodazole and were treated with UV-C (50 J/m2)inice- mutants, the chromosome restitution was clearly delayed further cold water. UV-irradiated and mock-treated controls then were plated to YPDA (∼2 h; Fig. 2 A and D), demonstrating a quantitative impact of medium in serial 10-fold dilutions. Survival was determined after 2 d of growth. the TLS polymerases. Although rad30Δ rev3Δ mutants had

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Fig. 2. TLS deficiency leads to the accumulation of ssDNA gaps and reduced repair efficiency. G2-arrested WT cells and TLS haploid mutants were treated with 50 J/m2 UV and were incubated in YPDA with nocodazole for up to 4 h (30 °C). Cells were collected to make DNA plugs and then were subjected to no MBN treatment (−MBN) (A) or to MBN treatment (+MBN) (B). (C) G2 cells were irradiated with 800 Gy (+IR) or were not irradiated (−IR). (D) Quantitation of SMD. After PFGE, chromosomes were visualized by ethidium bromide staining. In all figures, the letter “C” above the first lane stands for mock-treated control samples. The SMD is detected as a wide band of DNA between the largest chromosome (Chr XII) and the wells. The relative amounts of SMD were quantified using the ratio of the amount of material in the SMD region to the DNA in the small chromosomes that experienced little, if any, loss and are shown as “SMD/Chr I+VI”. Values shown in D are mean ± SD calculated from four independent experiments. Results shown in A and B are representative of multiple experiments.

additional delay, there was restitution of the chromosome bands unlike G1 cells (14). Consistent with a greater amount of SMD, by 4 h. the MBN revealed much more gapped DNA in the rad30Δ and Previous studies suggested that SMD appearing after UV ir- rad30Δ rev3Δ mutants during post-UV incubation. However, the radiation or MMS treatment likely results from long ssDNA restituted chromosomes at 4 h were less subject to MBN deg- formed during NER and possibly from recombination inter- radation, demonstrating that the gapped regions also could be mediates (9, 14). To establish the presence of ssDNA, we treated repaired in the TLS mutants. The repair appeared to be slower the agarose plugs containing chromosomal DNA with mung than in WT cells, as reflected by the somewhat greater MBN bean nuclease (MBN), which can degrade ssDNA tails and gaps degradation of the DNA from the TLS mutants at 2 and 4 h. This and cleave hairpin loops (described in ref. 18). Treatment with difference is consistent with the comparable survivals of WT cells MBN eliminated SMD and resulted in the appearance of frag- and the double-TLS mutants shown in Fig. 1. mented chromosomes (Fig. 2B) as found after exposure to ion- Because NER can repair most UV-induced damage, we asked izing radiation (Fig. 2C). These findings were similar to those in what role it might play in SMD formation in the irradiated G2 the study of Giannattasio et al. (14), who used S1 nuclease in G1 cells. We created rad14Δ rad30Δ or rad2Δ rad30Δ double mutants cells. Unlike S1 nuclease, which also acts on ssDNA nicks (26, that were deficient in NER. Rad14 is a homolog of human XPA 27), MBN is specific to ssDNA tails and gaps. Previously, we had protein, which forms a complex with ssDNA endonucleases addressed resection at random DSBs and found that duplex Rad1 and Rad10 and nicks the damaged DNA strand on the 5′ DNA with resected ssDNA tails of a few hundred to thousands side of a lesion (29, 30), whereas Rad2, a homolog of human of bases also exhibited retarded mobility on PFGE (referred to XPG, leads to incision at the 3′ side (31). Comparing the results as “PFGE-shift”) with a maximum apparent increase in molec- in Fig. 2 and Fig. 3A, we conclude that SMD formation is ular weight of 150 kb (18, 28). However, there was no evidence of downstream of NER. Furthermore, the chromosomal DNA from SMD or trapping of DNA in the well. Therefore, we concluded the double mutants, unlike that from the rad30 single mutant, that the SMD is the result of DNA repair intermediates con- was not sensitive to MBN (Fig. 3A). These findings also suggest taining ssDNA gaps. that there are no other mechanisms for the generation of gaps As shown in Fig. 2B, the MBN cutting had the greatest effect after UV treatment of the G2 cells in the absence of NER. on samples that had large amounts of SMD and had no effect on chromosomal DNA from unirradiated cells. Here, we also show HR Is Required to Repair UV Lesions in G2 Cells Lacking TLS. The that the restituted chromosomes from WT cells at 4 h were not finding that chromosomes still can be restituted after the ap- sensitive to MBN, suggesting complete repair of ssDNA gaps, pearance of SMD in the absence of TLS suggests an alternative

Ma et al. PNAS Early Edition | 3of10 Downloaded by guest on September 25, 2021 Fig. 3. SMD was prevented by blocking NER but was not affected by HR deficiency. G2/M-arrested WT cells and rad30Δ rad14Δ, rad30Δ rad2Δ, and rad51Δ mutants were treated with 50 J/m2 UV and were incubated in YPDA with nocodazole for up to 4 h (30 °C). Cells were collected to make DNA plugs. (A) After PFGE of DNA with or without MBN treatment, chromosomes were visualized by ethidium bromide staining. (B) After PFGE, chromosomes were visualized by ethidium bromide staining and Southern blot with a probe that identifies Chr III. Results shown in A and B are representative of multiple experiments.

efficient repair system(s) for dealing with the ssDNA, and such inactivation of both pathways did, we investigated the contribu- repair systems might explain the UV resistance of the TLS tion of HR to the appearance of SMD and the restitution of mutants (Fig. 1). Because deletion of either of the TLS genes chromosomes. REV3 or RAD30 or the HR genes RAD52 or RAD51 did not As shown in Fig. 4A, UV irradiation of double mutants de- markedly increase the sensitivity of G2 cells, but simultaneous fective for a TLS gene and an HR gene (i.e., rad30Δ rad51Δ or

Fig. 4. The redundancy of TLS and HR in repairing UV damage in G2. G2-arrested WT cells and HR and TLS haploid mutants were treated with 50 J/m2 UV and were incubated in YPDA with nocodazole for up to 4 h (30 °C). Cells were collected at the indicated times and processed for PFGE analysis. Chromosomes were visualized by ethidium bromide staining (A) and by Southern blotting (B) with a probe that identifies Chr III. Results shown are representative of multiple experiments.

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1301676110 Ma et al. Downloaded by guest on September 25, 2021 rad30Δ rad52Δ) results in the accumulation of SMD and eventual in the HR mutants, these results suggest that there is little gener- PNAS PLUS restitution of the chromosomes as seen in single mutants, although ation of DSBs. Previously, we had estimated that the background restitution may be somewhat delayed. (Deletion of RAD51 alone spontaneous level of DSBs in nongrowing cells is around one or two had no apparent effect on chromosome disappearance and res- DSBs per yeast genome (23). Based on a two- to three-fold in- titution when compared with WT cells, as shown in Fig. 3B.) A crease, at most, in band density after UV irradiation (Fig. 4, the 0.5- complete blockage of TLS and HR in the rad30Δ rev3Δ rad51Δ and 1-h lanes) over the background density (Fig. 4, lane “C”), the triple mutant also did not prevent the accumulation of SMD transient formation of DSBs resulting from exposure to 50 J/m2 UV (Fig. S2). However, the restitution of full-size chromosomes was is at most two to six DSBs per yeast genome. Given the experi- completely blocked (Fig. 4 and Fig. S2). The disappearance of mental variation, there appears tobelittledevelopmentofDSBs the chromosomes in the triple mutants may be caused by chro- from UV-induced lesions. mosomes being trapped in the well, because MBN treatment When the DNA plugs from UV-irradiated cells were treated generated fragmented chromosomes and a linear Chr III band with MBN, however, a large increase in the linear Chr III band (see ethidium bromide-stained and probed gels in Fig. S2). Also, was detected, especially at the 0.5- and 1-h time points (Fig. 5), if DNA is retained in the well, the amount of SMD appearing in corresponding to the time of the maximum appearance of SMD the gel (ethidium bromide staining) would not reflect all the molecules (Fig. 4A). This increase provides further evidence that SMD, thereby possibly explaining the apparently similar amounts many of the circular molecules contained ssDNA gaps. After of SMD in the rad30Δ rev3Δ rad51Δ and rad30Δ rev3Δ rad52Δ incubation, the ChrIII band densities of the WT and rad30Δ cells mutants and the TLS single mutants. Overall, these data suggest returned to the levels seen in unirradiated cells. However, some that HR is not involved in the generation of SMD but plays DSBs remained in the rad30Δ rad51Δ and rad30Δ rad52Δ strains a critical role in the downstream repair. HR is indispensable only (Fig. 4), as is consistent with the reduced levels of chromosome when both TLS polymerases are absent. The findings with the restitution at 2 and 4 h seen on ethidium bromide-stained gels UV-irradiated G2 cells are consistent with the greatly increased (Fig. 4 and Fig. S2). sensitivity of the triple mutants described in Fig. 1. ssDNA has long been suspected to be a component in re- combination (models are summarized in ref. 34). The Chr III Compromised TLS Activity Leads to Increased Recombinant Molecules and PFGE approach we used here also provides an opportunity After UV Irradiation. The requirement for HR in the absence of to detect recombination between sister chromatids. In our pre-

TLS could be caused by the generation of DSBs, possibly vious study we had detected the appearance of a double-sized GENETICS through overlapping repair regions as previously suggested (32, chromosome III (Chr III dimer) band on PFGE in G2 cells ex- 33) or somehow during the generation of the ssDNAs. Using our posed to ionizing radiation (18). This Chr III dimer can be previously described circular chromosome assay that can detect formed only when two sister chromatids combine with each random DSBs efficiently at frequencies of only a few DSBs per other and thus provides an unambiguous way to detect inter- yeast genome (18, 23), we investigated whether TLS deficiency chromosome recombination. As shown in Fig. 4, a similar dimer leads to increased DSBs during the processing of UV damage. In band was not apparent after UV irradiation. However, treatment this assay, a circular Chr III, which is ∼300 kb, is retained in the of the DNA sample with MBN revealed the presence of dimer well during PFGE. A random DSB will generate a linear chromo- molecules (Fig. 5 and Fig. S2). Because MBN cuts at ssDNA some that enters the gel and forms a distinct band during PFGE. gaps, these molecules must have come from circular Chr III As shown in Fig. 4, there was at most only a small induction of dimers or the intermediates of recombination between the Chr DSBs following UV exposure of WT cells, based on the limited III sister chromatids The recombinant product was clearly detect- increase in linear Chr III molecules. The amounts of linear Chr able by 1 h after irradiation of G2 WT cells, reaching a maximum III bands were similar to those in the rad30Δ, rad30Δ rad51Δ and 2–4 h postirradiation. Although the absolute number of Chr III rad30Δ rad52Δ mutants. Because DSBs are expected to accumulate dimers could not be determined, the relative difference in dimer

Fig. 5. Compromised TLS function leads to the generation of recombinants. G2-arrested WT cells and HR, TLS, and NER haploid mutants were treated with 50 J/m2 UV and were incubated in YPDA with nocodazole for up to 4 h (30 °C). Cells then were collected to make DNA plugs and were treated with MBN. Chromosomes were visualized by ethidium bromide staining (A and C) and by Southern blotting (B and D) with a CHA1 probe that identifies Chr III. Linear Chr III and Chr III dimers (as described in ref. 18) are indicated. Results shown are representative of multiple experiments.

Ma et al. PNAS Early Edition | 5of10 Downloaded by guest on September 25, 2021 bands across the same gels could be compared based on band To address the relationship between DSB induction and re- density. (Sample loading was comparable, as determined by the combination, we compared the efficiency of DSB induction with chromosome bands in the ethidium bromide-stained gels of the the formation of recombinants after exposure of the G2 cells to smallest chromosomes, Chr I and VI, which experienced little ionizing radiation and UV. The WT and rad30Δ G2 cells were change during incubation, as shown in Fig. 5 and Fig. S2.) We irradiated with 400 Gy or 50 J/m2. After irradiation and in- found that by 2 h after irradiation the rad30Δ cells had approx- cubation, samples were split and treated with MBN to determine imately three times more Chr III dimers than the WT cells (Fig. the gapped molecules generated in vivo and the generation of S2). The amount of dimer molecules appears to be related to the Chr III dimer molecules. As shown in Fig. 6, a large number of amount of ssDNA. The Chr III linear band generated after MBN DSBs generated by ionizing radiation subsequently were lost treatment was approximately two- to three-fold greater for the through repair. In these experiments there was only a small in- rad30Δ cells than for the WT cells. Formation of the Chr III crease in broken molecules during post-UV incubation. How- dimer was detected after SMD began to appear. It is possible ever, despite a greater than 20-fold difference in DSBs between that completed recombinants lacking ssDNA gaps are generated cells exposed to ionizing radiation and UV-irradiated cells, there also, but these molecules would remain in the well after MBN were comparable amounts of recombinant molecules in the WT cells. For the rad30Δ cells, there were nearly three-fold more treatment. Because there was no dimer band in the rad30Δ rad51Δ recombinants after UV irradiation than after exposure to ion- and rad30Δ rad52Δ double mutants, the generation of recombi- izing radiation. The time frame for the generation of recombi- nants requires RAD52 and RAD51. nants by exposure to ionizing radiation and UV irradiation was Overall, these results establish that recombinant molecules are comparable despite the immediate formation of DSBs in the generated during incubation after UV treatment of G2 cells and cells exposed to ionizing radiation. If DSBs were the source of that reduced TLS leads to more recombinants. We note that for recombinant molecules, there should be far fewer recombinants the rad30 rev3 double TLS mutant there was no apparent in- rad30 after UV irradiation, contrary to our observations. Thus, DSBs crease of Chr III dimer over that in (Fig. S2). Possibly do not seem to be the source of recombinant molecules. there is an adverse effect of rev3 deletion or the loss of both TLS polymerases on the recombination process (35–37). UV-Induced Recombination Requires Gap Expansion. Recently, Gian- nattasio et al. (14) showed that the ssDNA gaps formed after UV Recombinant Molecules Do Not Correlate with DSBs. The increase in irradiation in G1 yeast cells could be expanded by Exo1 to up to B recombinant molecules following MBN treatment (Fig. 5 ) did 500 nt, and the resulting ssDNA led to checkpoint activation. We not appear to correlate with the few DSBs (Fig. 4B) observed deleted EXO1 in a rad30Δ mutant to examine whether the gen- with the WT cells or the TLS mutants, which had more ssDNA eration of Chr III dimers after UV was dependent on large gaps. However, the recombination required NER, as shown by stretches of ssDNA. As shown in Fig. 7, there was a nearly 2-h the absence of Chr III dimers after MBN treatment of the UV- delay in the appearance of ssDNA based on the formation of irradiated rad14Δ rad30Δ and rad2Δ rad30Δ double mutants SMD in cells not treated with MBN. Similarly, the appearance of (Fig. 5D). These results could be explained by a recombination Chr III recombinants was delayed in cells treated with MBN and mechanism initiated by ssDNA gaps or even by nicks rather there were fewer recombinant molecules in the rad30Δ exo1Δ than DSBs. strain than in the rad30Δ strain. These results indicate that

Fig. 6. Comparison between ionizing radiation and UV for the induction of DSB and generation of recombinants. G2-arrested WT cells (A) and rad30 (B) haploid mutants were treated either with 400 Gy ionizing radiation (IR, Left) or 50 J/m2UV (Right) and then were incubated in YPDA with nocodazole for up to 4 h (30 °C). Cells were collected to make DNA plugs and were processed further with or without MBN treatment, followed by PFGE and Southern blotting to detect Chr III linear, resected linear, and recombinant dimer molecules, as indicated.

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Fig. 7. exo1 delays the appearance of both SMD and Chr III dimer. G2-arrested rad30 and rad30 exo1 haploid mutants were treated with UV (50 J/m2)and were incubated in YPDA with nocodazole for up to 4 h (30 °C). Cells then were collected to make DNA plugs and were processed with or without MBN treatment. Chromosomes were visualized by ethidium bromide staining and by Southern blotting with a CHA1 probe that identifies Chr III linear, resected, and recombinant dimer molecules, as indicated.

recombination after UV exposure requires gap expansion to long support of this view, we have observed a dramatic difference ssDNA from the initial NER excision tract of ∼30 nt and is pro- between WT cells and TLS mutants including (i) elevated for- vided largely by Exo1. mation of SMD in rad30 or rev3 mutants along with more SMD in the double mutant and (ii) a greater amount of linear Chr III Discussion band in TLS mutants after in vitro MBN treatment that converts ssDNA gaps are potentially genome destabilizing. If unrepaired, gaps into DSBs. Even when TLS is functioning in WT cells, there they can lead to DSBs during subsequent replication. In addi- is a small amount of SMD. This SMD may be caused by in- tion, ssDNA is more susceptible to mutagens because there is no sufficient TLS proteins, by some types of lesions not being effi- complementary strand to template the correction of lesions (38). ciently bypassed, or, occasionally, by efficient Exo1-mediated gap Here, we show that the combined three systems available in most expansion before TLS recruitment. In any case, we have shown eukaryotes to provide toleration of bulky lesions—NER, TLS, that SMD results from long ssDNA gaps (or from some sort of and HR—prevent and repair ssDNA that can arise in G2 cells. downstream repair intermediates) in UV-irradiated G2 cells and The HR-mediated repair appears to act directly on the ssDNA that the formation of SMD is not Rad51 or Rad52 dependent. A and, unexpectedly, not through DSB intermediates. The repair related gap-expansion phenomenon also was observed in bacte- of the ssDNAs would allow cells to proceed into the next cell ria. Earlier studies showed that, although the majority of UV cycle. In the absence of ssDNA repair, DSBs can appear in the lesions in Escherichia coli result in short repair patches of ∼20–30 nt, subsequent round of replication, which may explain the UV a small proportion leads to long patches of up to several hundred sensitivity of TLS/HR double mutants irradiated in G2. nucleotides (42). In addition, mutants deficient in polymerase I The filling of the excised region during NER is highly co- or its 5′ exonuclease activity led to more long patches that were ordinated with the excision steps so that normally there are only proposed to be due to gap expansion by a 3′ exonuclease and short-lived small ssDNA intermediates (11). In bacteria, the resynthesis by DNA polymerases II or III (42, 43). The poly- damaged oligonucleotide is removed by the UvrD just merases II and III had been shown to generate longer patches before or during the repair synthesis step by DNA polymerase I than polymerase I (44). (39). The accumulation of ssDNA in TLS mutants after UV ir- Based on an earlier study, irradiation with 50 J/m2 UV-C (254 radiation of G2 cells could result from closely opposed lesions. nm) would yield ∼0.5 pyrimidine dimers/kb (25). If NER gen- After NER of one of the lesions, the subsequent repair synthesis erates ∼30-nt excised regions (3) that are distributed randomly, would encounter the second lesion, which could be bypassed by approximately 0.004 closely opposed lesions (within 30 nt of each TLS polymerases (described in Fig. 8). Consistent with this re- other) would be expected per kilobase. The frequency actually sult, the error-prone Y family DNA polymerase Pol κ has been may be larger, because the appearance of closely opposed lesions shown to have an important role in NER for repair synthesis in does not follow dose-squared kinetics, at least at lower doses mammalian cells (40, 41). Apart from its other functions, this (32). Thus, yeast chromosomes with a length of around 300 kb polymerase might be used to deal with closely opposed DNA would contain an average of approximately one pair of closely lesions. The absence of TLS may result in small gaps being en- opposed lesions that might be expected to require damage by- larged by Exo1, leading to SMD, as shown previously for cells pass during repair. This estimate is reflectedinourPFGE arrested in G1 (14) and also found for G2 cells in this study. In results, in which the disappearance of chromosomes was size

Ma et al. PNAS Early Edition | 7of10 Downloaded by guest on September 25, 2021 known whether such lesion-containing gaps were reparable only during S phase or could be processed in nonreplicating stages. The repair of ssDNA gaps in G2 cells appeared to occur through an HR mechanism that is independent of DSB forma- tion. Lesions other than DSBs, including ssDNA breaks (SSBs), have been proposed as initiators of recombination in many early models of recombination (34). More recently, an SSB was shown to initiate gene conversion to generate mating-type switching in fission yeast (48). Also, recombination-activating gene (RAG)– mediated nicks efficiently stimulate HR during V(D)J re- combination (49). In other studies using enzymes that generate site-specific nicks, SSBs could promote recombination (50, 51). However, in many of these studies it was not clear whether HR was initiated by SSBs per se or by subsequent formation of DSBs. Using an assay that directly measures recombination by the appearance of Chr III dimer molecules from sister chromatids, we have concluded that ssDNA can initiate HR in nonreplicating G2 cells. First, the formation of Chr III dimers required Rad51/ 52-mediated HR. Second, there was no correlation between the number of DSBs and the appearance of Chr III dimers. Many more chromosome dimers were produced with exposure to UV than by exposure to ionizing radiation, although ionizing radia- tion produced vastly more DSBs. In addition, there is no cor- relation between the timing of DSB induction and Chr III dimer formation. Although ionizing radiation directly induces DSBs, followed within several minutes by repair-related resection, Chr III recombinant molecules appeared at about the same time after gamma and UV irradiation (∼1 h; Fig. 6). That ssDNA is required for at least the initiation of recombination comes from the observations that both NER and ExoI are needed for the generation of SMD as well as recombinants and that deficiencies in TLS, which greatly increase SMD, lead to corresponding increases in recombinant molecules. Although the mechanism by which an ssDNA gap could trig- ger sister chromatid recombination remains to be determined, the ssDNA gaps directly formed by the NER excision step are Fig. 8. Model for ssDNA gap induction of recombination. After UV irradi- insufficient to activate HR. Deletion of EXO1 greatly reduced ation, the processing of closely opposed lesions by NER could generate a gap the amount of ssDNA as well as recombinant molecules (Fig. 7). opposite a UV lesion, which is normally tolerated by TLS. In the absence of Exo1 is a structure-specific nuclease possessing both 5′–3′ exo- TLS or when the gap is not efficiently refilled, it is subject to gap expansion nuclease and 5′-flap endonuclease activity and is involved in by Exo1. The resulting gap or the stalled nascent strand can lead to re- a variety of DNA metabolic processes (52). Previous results with combinational interactions, including strand invasion to generate recombi- nant intermediates. The homologous sequence in the sister chromatid serves G1 cells demonstrated that Exo1-extended gaps can lead to the as a template allowing the gap opposite the lesion to be filled. activation of a cellular checkpoint (14). Possibly, additional events following Exo1-dependent checkpoint activation also can trigger HR repair. Also, the expanded gaps might participate dependent; irradiation with 50 J/m2 UV led to no apparent loss directly in a pathway that initiates recombination, similar to the of the smallest chromosomes, Chr I and VI (∼200 kb each). Our recombinational role that resection plays at DSBs. findings with Chr III suggest that there is little conversion of Fig. 8 presents a model that summarizes our findings and closely opposed lesions into DSBs, possibly indicating repair describes how ssDNA gaps could be channeled to HR for repair interference, unlike the situation in closely opposed MMS- when TLS is defective. Although we have established several of induced lesions (8). the parameters required for the generation of recombination, the In this study, we establish that HR can repair the ssDNA gaps mechanisms of interactions between sister chromatids and the that arise in G2 cells when TLS is defective, thereby acting as actual exchange events remain to be determined. After ssDNA a back-up in gap repair. Contrary to findings in G1 cells, which formation, subsequent step(s) in recombination require homol- ogy search and might include pairing of the ssDNA with the lack the opportunity for recombinational repair (14), in the G2 homologous double-strand region of the sister chromatid or lo- cells we found efficient loss of SMD at later times, restitution of calized unwinding of the blocked nascent strand and homology chromosomes, the disappearance of MBN-sensitive sites, and search as has been suggested for template switching at uncoupled high survival rates, all of which required Rad52 and Rad51. replication forks (53). Regardless of the initiating scenario, the Although recombination repair can restore collapsed replication subsequent events are expected to include strand invasion and forks (45, 46), there is little direct evidence that HR directly synthesis from the sister chromatid DNA. Given our recent processes and repairs replication-blocking lesions located on findings that sister chromatid cohesion has a role in preventing ssDNA. Using a plasmid-based assay, Adar et al. (47) showed DSB-induced recombination between homologous chromosomes that gaps opposite an abasic site or a benzo[a]pyrene-guanine (54), it would be interesting to determine whether this cohesion adduct could be repaired by HR in mammalian cells. However, effect extends to ssDNA. it was not clear whether the lesions on the plasmid were direct Although the current study used recovery after UV irradiation as substrates for recombination repair or were converted into other a model for repair of bulky lesions and recombination, the mecha- types of HR-responsive lesions in vivo. In addition, it is not nism for the generation of ssDNA and the efficient recombinational

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1301676110 Ma et al. Downloaded by guest on September 25, 2021 repair of gaps likely is the same for a variety of bulky lesions. These plugs then were incubated with a “spheroplasting” solution [1 M sorbitol, PNAS PLUS findings, which are expected to expand our understanding of mech- 20 mM EDTA, 10 mM Tris (pH 7.5)] for 2 h followed by digestion with pro- anisms of HR and the complementary relationship between TLS and teinase K [10 mM Tris (pH 8.0), 100 mM EDTA, 1.0% N-lauroylsarcosine, 0.2% HR, have direct implications for genome stability in other eukaryotes, sodium deoxycholate, 1 mg/mL proteinase K] for 24 h at 30 °C. Yeast chro- including humans. mosome DNA was separated by PFGE using a Bio-Rad CHEF-Mapper XA system (Bio-Rad) on 1% agarose gel (6 V/cm for 24 h at 14 °C, 10–90 s ramp Materials and Methods and 120° switching angle). Gels were stained with ethidium bromide, and fi Yeast Strains. Yeast strains used in this study are derivatives of two isogenic Southern blot analysis was carried out with a probe speci c for Chr III. haploid strains, MWJ49 and MWJ50 (MATα leu2-3,112 ade5-1 his7-2 ura3Δ Autoradiographs were digitized and densitometric analysis was performed trp1-289), which contain a circular form of Chr III (23). The deletion mutants using Kodak MI software (version 5.0). Comparison of the relative chromo- rad30, rev3, rad50, , rad52, exo1, and their derived double or triple some band densities of different mutants was done within the same mutants were created by replacing the relevant ORF in these strains with Southern blot. The comparable amounts of DNA loading were judged based selectable markers as previously described (55). on ethidium bromide staining. Note: Comparisons between different blots are not accurate because of variations in Southern transfer and radioactive UV and Ionizing Irradiation. Haploid yeast cells were synchronized in G2 with signals. The PFGE results that are presented in the figures are representative nocodazole (15 μg/mL) as described in ref. 9; the extent of arrest is described of at least two gels. in Table S1. For UV irradiation, cells in ice-cold water were exposed to 2 2 50 J/m UV-C [254 nm, 0.85 J/(m ·s)] using cold cathode model 782L10 lamps MBN Digestion. MBN (Promega) digestion of DNA in agarose plugs was used from American C). Post-UV incubation in nocodazole is described to cut/degrade ssDNA regions formed during the repair of UV-induced 137 in Table S1. For ionizing irradiation, cells kept on ice were irradiated in a Cs damage. A 50-μL plug slice was equilibrated at room temperature in 150 μL irradiator (J. L. Shepherd Model 431) at a dose rate of 2.3 krad/min to a of TE [10 mM Tris (pH 7.4), 1 mM EDTA], followed by 30-min incubation with total dose of 400 Gy. After irradiation, cells were returned to YPDA me- 80 units/mL MBN (controls were incubated in reaction buffer without en- dium [1% yeast extract, 2% (wt/vol) Bacto-Peptone, 2% (wt/vol) dextrose, 60 zyme) and then equilibration with ice-cold Tris-EDTA [10 mM Tris, 50 mM mg/mL adenine sulfate] containing nocodazole (G2) and were incubated for EDTA (pH 8.0)] to stop the reaction. PFGE Southern blot analysis was per- up to 4 h (30°C). Cells were collected at various times before and after ir- formed as described above. radiation for analysis by PFGE and Southern blot probing.

ACKNOWLEDGMENTS. We thank Drs. Julie Horton, Dmitry Gordenin, Shay PFGE Analysis. Detection of UV-induced damage and repair were based on fl Covo, and Kin Chan for critical reading of the manuscript and suggestions. PFGE analysis as previously described (23). Brie y, control and UV-treated This work was supported by the Intramural Research Program of the GENETICS cells were mixed with low-melting agarose [2% (wt/vol) stock in Tris-EDTA National Institute of Environmental Health Sciences (National Institutes buffer, final concentration 0.6%] containing 1 mg/mL Zymolyase (100 U/mg) of Health, Department of Health and Human Services) under Project 1 Z01 (MP Biochemicals) to prepare agarose-embedded DNA plugs. The cells in the ES065073 (to M.A.R.).

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