Requirement of Rad18 Protein for Replication Through DNA Lesions in Mouse and Human Cells

Requirement of Rad18 protein for replication through DNA lesions in mouse and human cells

Jung-Hoon Yoon, Satya Prakash, and Louise Prakash1

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX 77555

Edited* by Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved March 21, 2012 (received for review March 8, 2012) In yeast, the Rad6-Rad18 ubiquitin conjugating enzyme plays a translocase activity that could function directly in lesion bypass critical role in promoting replication although DNA lesions by by promoting replication fork regression and template switching translesion synthesis (TLS). In striking contrast, a number of (11). Genetic studies in yeast have provided evidence for the studies have indicated that TLS can occur in the absence of requirement of Mms2, Ubc13, and Rad5 for a template-switch- Rad18 in human and other mammalian cells, and also in chicken ing mode of lesion bypass and they have shown that both the cells. In this study, we determine the role of Rad18 in TLS that ubiquitin ligase and DNA translocase activities are essential for occurs during replication in human and mouse cells, and show that Rad5 to carry out its role in lesion bypass (6, 12). in the absence of Rad18, replication of duplex plasmids containing Mammalian cells have two RAD6 homologs, RAD6A and a cis-syn TT dimer or a (6-4) TT photoproduct is severely inhibited RAD6B, and a single RAD18 gene. Importantly, in contrast to in human cells and that mutagenesis resulting from TLS opposite the indispensability of the Rad6-Rad18 enzyme and of PCNA cyclobutane pyrimidine dimers and (6-4) photoproducts formed at ubiquitylation for TLS in yeast cells, several studies have in- the TT, TC, and CC dipyrimidine sites in the chromosomal cII gene dicated that Rad6-Rad18 and PCNA ubiquitylation may not play in UV-irradiated mouse cells is abolished. From these and other as significant a role in TLS in vertebrates as in yeast. For ex- observations with Rad18, we conclude that the Rad6-Rad18 en- ample, in two studies carried out with human cell-free extracts, zyme plays an essential role in promoting replication through TLS was shown to occur in the absence of PCNA ubiquitylation. DNA lesions by TLS in mammalian cells. In contrast, the dispens- In one study, replication through a site-specific cis-syn TT dimer ability of Rad18 for TLS in chicken DT40 cells would suggest that present on the leading-strand template of a double-stranded GENETICS the role of the Rad6-Rad18 enzyme complex has diverged consid- circular plasmid was examined in human cell-free extracts using erably between chicken and mammals, raising the possibility that conditions in which origin-dependent initiation and bidirectional TLS mechanisms differ among them. replication occur. In this system, replication through the TT dimer carried on the plasmid is absolutely dependent upon Polη; human Rad6-Rad18 enzyme | lesion bypass | UV damage however, when replication opposite the dimer was examined with cell-free extracts using wild-type PCNA or K164R mutant PCNA NA lesions in the template strand block the progression of defective in ubiquitylation, TLS by Polη could occur with K164R Dthe replication fork. Genetic studies in the yeast Saccharo- mutant PCNA (13). In another study, TLS opposite a cis-syn TT myces cerevisiae have indicated a critical role for the Rad6-Rad18 dimer carried on a single-stranded plasmid was analyzed using ubiquitin-conjugating complex (1, 2) in promoting replication human cell-free extracts that lacked Rad18 or in which the through DNA lesions by translesion DNA synthesis (TLS) and K164R mutant PCNA was used instead of wild-type PCNA, and by an alternative pathway that involves template switching (3). TLS was shown to occur in the absence of Rad18 as well as in the In yeast, during replication, TLS through UV-induced cis-syn absence of PCNA ubiquitylation (14). cyclobutane pyrimidine dimers (CPDs) occurs by the action of In another study, TLS has been examined in mouse cells − − DNA polymerases (Pols) η and ζ, in which Polη performs rela- lacking Rad18 (Rad18 / ) using a gapped plasmid that carried tively error-free synthesis opposite CPDs, whereas Polζ carries a site specific cis-syn TT dimer, a (6-4) TT photoproduct, or a cis- out a more mutagenic mode of TLS (3, 4). Consequently, the Pt GG intrastrand crosslink (15). Opposite all three DNA − − incidence of UV-induced mutations is elevated in rad30Δ cells lesions, TLS occurred with a frequency of ∼30–40% in Rad18 / lacking Polη (5) and is greatly reduced in yeast cells lacking Polζ cells compared with that in wild-type cells; and opposite each of (3). A Rad5-Mms2-Ubc13–dependent template-switching path- these DNA lesions, the relative frequency of error-free and − − way provides an error-free alternative to allow for the passage of mutagenic TLS remained the same in Rad18 / cells as in wild- the replication fork through DNA lesions (6). Because of the key type cells. Thus, although Rad18 affected the efficiency of TLS, role of Rad6-Rad18 in advancing replication through DNA mutagenicity was not affected. Taken together, the above-noted lesions, yeast cells lacking Rad6 or Rad18 protein exhibit a very studies have supported the inference that in mammalian cells, high degree of sensitivity to DNA damaging agents, and because TLS Pols can function in lesion bypass in the absence of Rad6- of the requirement of this enzyme complex for both the error-free Rad18 enzyme function. and error-prone modes of TLS, mutagenesis induced by DNA Studies with chicken B lymphocyte DT40 cells have also in- damaging treatments is inhibited in rad6Δ/rad18Δ yeast cells (3). dicated that TLS can occur in the absence of Rad18. For ex- Treatment of yeast cells with DNA damaging agents elicits ample, although a deficiency of either Polκ or Rad18 confers an monoubiquitylation of proliferating cell nuclear antigen (PCNA) increase in UV sensitivity in DT40 cells, they both impart about at lys164 by the Rad6-Rad18 enzyme; subsequently, this PCNA the same level of UV sensitivity; and an additive increase in UV residue is polyubiquitylated via a lys63-linked polyubiquitin chain, for which the Mms2-Ubc13-Rad5 complex is additionally required (7). Genetic studies in yeast have shown that PCNA Author contributions: J.-H.Y., S.P., and L.P. designed research; J.-H.Y. performed research; monoubiquitylation is a necessary prerequisite for TLS and that J.-H.Y., S.P., and L.P. analyzed data; and J.-H.Y., S.P., and L.P. wrote the paper. Rad6-Rad18–dependent template switching requires PCNA The authors declare no conflict of interest. polyubiquitylation (7–9). In the Mms2-Ubc13-Rad5 complex, the *This Direct Submission article had a prearranged editor. Mms2-Ubc13 ubiquitin conjugating enzyme (10) functions in 1To whom correspondence should be addressed. E-mail: [email protected]. conjunction with Rad5 protein, which provides the ubiquitin li- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gase function (7). Rad5 also possesses a DNA helicase/DNA 1073/pnas.1204105109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1204105109 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 sensitivity occurs in the absence of both Polκ and Rad18 (16). An siRNA-treated XPA cells, Rad18 depletion led to a 65% re- important implication of these observations is that Rad18 is duction in UV survival, whereas UV survival of Polη as well as of not essential for TLS and that Polκ can function in TLS in- Polζ (Rev3)-depleted cells was reduced by ∼30%. In accord with dependently of Rad18. If Rad18 were indispensable for TLS by a less significant role of Polκ in TLS, UV survival was impacted the various Pols involved in the bypass of UV lesions, then Rad18 the least upon Polκ depletion. Importantly, we find that simul- deficiency would have conferred a much higher UV sensitivity taneous depletion of Rad18 with any of the TLS Pols, η, ζ,orκ, than that resulting from a deficiency of Polκ or of any other in- conferred no further reduction in UV survival than that seen dividual Pol involved in TLS opposite UV lesions, and there would upon depletion of Rad18 alone. Hence, an epistatic relationship have been no further increase in UV sensitivity of Rad18 deficient exists between Rad18 and any of the TLS Pols that are required cells in combination with mutations in any of the TLS Pols. for replicating through UV lesions. In another study, the role of Rad18 and of PCNA ubiqui- We also verified the epistasis of Rad18 with TLS Pols in tylation was analyzed in UV-irradiated or 4-nitroquinoline-1- mouse cells. As shown in Fig. S2B, compared with control (NC) oxide (NQO)-treated chicken DT40 cells by DNA fiber labeling siRNA-treated cells, Rad18 depletion conferred an ∼60% re- (17). Rather surprisingly, the rate of fork progression through duction in UV survival, depletion of either Polη or Polζ resulted damaged DNA was not affected in rad18 mutant cells or in cells in ∼30% reduction in UV survival, whereas simultaneous deple- carrying the K164R mutation in PCNA. Furthermore, disruption tion of Rad18 with any of the TLS Pols conferred no further of Rev1 TLS Pol resulted in a reduction in the rate of fork reduction in UV survival over that observed in Rad18-depleted progression through DNA lesions, and sensitivity to UV or NQO cells. The epistatic interaction of Rad18 with TLS Pols would was greatly enhanced in DT40 cells carrying the rev1 mutation in suggest that both in human and mouse cells, Rad18 functions in combination with the K164R PCNA mutation. These observations conjunction with TLS Pols in lesion bypass. have added strong support to the notion that the Rad6-Rad18 enzyme and PCNA ubiquitylation are not as indispensable for Requirement of Rad18 for Replication of Plasmids Containing a cis- TLS in chicken cells as in yeast, and that in chicken cells TLS Pols syn TT Dimer or a (6-4) TT Photoproduct. To determine the role of can carry out lesion bypass in the absence of Rad6-Rad18. Rad18 in TLS, we examined the frequency of TLS in Rad18- Because in yeast cells, the Rad6-Rad18 complex and PCNA depleted human XPA cells opposite a cis-syn TT dimer or a (6-4) ubiquitylation play a key role in lesion bypass by TLS, and be- TT photoproduct carried in the leading strand template of cause replication through DNA lesions is greatly inhibited in a duplex plasmid where bidirectional replication initiates from the absence of Rad6 or Rad18 (3, 18), it becomes imperative to an SV40 origin (Fig. S3). In this plasmid system, TLS through the know whether during replication, TLS can occur in human cells DNA lesion results in a blue colony and because the lesion- + in the absence of Rad6-Rad18 or whether the Rad6-Rad18 en- containing DNA strand carries the wild-type Kan gene (Fig. zyme is as indispensable for TLS in humans as it is in yeast. To S3B), the frequency of TLS is determined from the number of + establish whether or not the Rad6-Rad18 enzyme plays an es- blue colonies among the total Kan colonies. Hence, the siRNA sential role in promoting replication through DNA lesions by knockdown of Pols required for TLS opposite the DNA lesion TLS Pols in mammalian cells, in this study we examine in human results in a reduction in the frequency of blue colonies. With the cells the effects of Rad18 depletion on the replication of a duplex undamaged plasmid, however, we observe no significant change + plasmid harboring a cis-syn TT dimer or a (6-4) TT photoproduct, in the frequency of blue colonies among Kan colonies, regard- and we analyze the effects of Rad18 depletion on mutagenesis less of which TLS Pols have been depleted; thus, the depletion of resulting from TLS opposite CPDs and (6-4) photoproducts TLS Pols affects only the frequency with which the lesion-con- formed in UV-irradiated mouse cells. From these studies in hu- taining DNA strand replicates, but has no affect on the repli- man and mouse cells, we conclude that Rad18 is indispensable for cation of undamaged plasmid. TLS in mammalian cells. We discuss the implications of our In a number of different experiments in Rad18-depleted cells, + observations for the role of Rad6-Rad18 enzyme complex and of we observed a dramatic reduction in the total number of Kan PCNA ubiquitylation in lesion bypass during replication in colonies when the plasmid carried a cis-syn TT dimer or a (6-4) + human cells. TT photoproduct, whereas the numbers of Kan colonies that could be recovered from the replication of undamaged plasmid Results were not affected in Rad18-depleted cells. These observations Epistasis of Rad18 with TLS Pols. A major role of Rad18 in TLS suggested that the absence of Rad18 has a profound effect on predicts that the absence of Rad18 would confer a large increase the replication of the damaged plasmid but not on the replica- in UV sensitivity and that the UV sensitivity of rad18 mutant tion of the undamaged plasmid. To verify these observations, we cells would not increase upon inactivation of any of the TLS cotransfected the lesion-containing plasmid together with an Pols. We have shown previously that in human and mouse cells, undamaged pCDNA 3.1.zeocin plasmid and with Rad18 siRNA Pols η, ζ, and κ provide alternate pathways for replicating into XPA cells that had been pretreated with Rad18 siRNA (Fig. through CPDs, the major UV photoproduct, in which Polη car- S3C). Hence, with these two plasmids, the replication efficiency ries out highly error-free TLS, and Pols ζ and κ contribute to of lesion-containing plasmid relative to the replication efficiency mutagenic TLS (19). For replicating through the less-frequently of undamaged plasmid can be determined from the relative + formed UV photoproduct, (6-4) PPs, Polζ functions in a pre- frequency of Kan colonies among zeocin-positive colonies. dominantly error-free pathway, whereas Polη and Polι provide The replication of undamaged plasmid was not affected by the alternate pathways of mutagenic TLS (20). To determine if an siRNA knockdown of Rad18 or of any of the TLS Pols, as the epistatic relationship exists between Rad18 and TLS Pols, we numbers of undamaged plasmid recovered were about the same examined the UV sensitivity of human XPA cells, defective in regardless of whether cells were treated with control siRNA or nucleotide excision repair (NER), that have been treated with with siRNAs for Rad18 or TLS Pols (Table 1). In XPA cells siRNAs to deplete either Rad18 or any of the TLS Pols known to treated with control (NC) siRNA, opposite both the cis-syn TT have a role in TLS opposite UV lesions, or where Rad18 was dimer and (6-4) TT photoproduct, TLS occurs with a frequency depleted in combination with depletion of one of the TLS Pols. of ∼35% (Table 1), and sequence analyses of blue colonies has The high efficiency of Rad18 depletion was verified by RT-PCR shown that mutations occur only opposite the 3′ T or the 5′ Tof and by Western blotting (Fig. S1), and the evidence for high either photoproduct (19, 20). In addition, from sequence anal- efficiency siRNA depletion of TLS Pols has been provided be- yses of over 300 white KanR colonies from control NC siRNA- fore (19). As shown in Fig. S2A, compared with control (NC) treated XPA cells, we have determined that they all contain the

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1204105109 Yoon et al. Downloaded by guest on September 27, 2021 Table 1. Effect of siRNA depletion of Rad18 and TLS Pols on the replication efficiency of plasmid carrying a cis-syn TT or a (6-4) TT photoproduct on the leading strand DNA template in NER-defective XPA human fibroblast cells Undamaged Lesion containing plasmid plasmid Replication efficiency of + No. of blue colonies No. of zeocin lesion-containing plasmid + + + + DNA lesion siRNA No. of Kan colonies among Kan TLS (%) colonies (Kan /zeocin )*

NC 658 228 34.7 642 1.02 cis-syn Polη 458 80 17.5 628 0.73 TT dimer Polη + Rev3 372 44 11.8 656 0.57 † † Rad18 65 — — 608 0.11 NC 612 203 33.2 608 1.00 (6-4)TT Rev3 451 70 15.5 625 0.72 photoproduct Polη + Rev3 302 38 12.6 643 0.47 † † Rad18 76 — — 614 0.12

*The experiments for determining the replication efficiency of lesion-containing plasmid were repeated five times and similar results were observed in different experiments. The data shown are from one representative experiment. † Because of the large reduction in the recovery of lesion-containing plasmid in Rad18-depleted cells, and because only a few blue colonies were observed in each experiment, we could not reliably estimate the TLS frequency for each individual experiment. However, when all of the experiments are considered together, we estimate that TLS frequency was reduced to ∼5%, which likely reflects the residual level of Rad18 that remains in siRNA-depleted cells.

SpeI sequence and that no other flanking mutations are present. To analyze mutagenesis resulting specifically from TLS op- Thus, any mutations that occur during the replication of dam- posite CPDs, the (6-4) photoproducts were removed from the aged plasmid are restricted only to the lesion site, indicating genome by expressing a (6-4) photolyase gene in BBMEF cells that the blue/white colony assay provides a good measure of using the experimental protocol that allows for the complete TLS frequencies. removal of (6-4) photoproducts from UV-irradiated cells (21). GENETICS As shown in Table 1, with the TT dimer-containing plasmid, As shown in Table 2, the frequency of mutations in unirradiated + − the frequency of Kan colonies relative to zeocin-positive colo- cells treated with control (NC) siRNA was ∼13 × 10 5, and nies was reduced by ∼25% upon knockdown of Polη alone and spontaneous mutation frequency was not affected in Rad18-de- by ∼40% upon knockdown of both Polη and Polζ. Rad18 depleted cells. In UV-irradiated mouse cells expressing the (6-4) pletion, however, led to a much more drastic reduction in the photoproduct photolyase gene and exposed to photoreactivating − replication of lesion-containing plasmid, as indicated from the light, the mutation frequency increased to ∼48 × 10 5. Because ∼ Kan+ recovery of only 10% colonies relative to zeocin-positive the (6-4) photoproducts have been removed with the experi- colonies. These effects of Rad18 depletion on the replication mental protocol being used, this mutation frequency represents fi − of undamaged and damaged plasmids have been veri ed in a the contributions of spontaneous mutations (∼13 × 10 5) plus − number of different experiments (Table 1). Similar results were those resulting from mutagenic TLS opposite CPDs (∼35 × 10 5). obtained with (6-4) TT photoproduct-containing plasmid. Rela- + tive to zeocin-positive colonies, the frequency of Kan colonies was reduced by ∼30% in Rev3-depleted cells, and by ∼50% in Table 2. Effects of Rad18 depletion on UV induced mutation cells depleted for both Polη and Rev3, whereas in Rad18 depleted frequencies in the cII gene in mouse cells (BBMEF) expressing + cells, the recovery of Kan colonies was reduced by ∼90%. Hence, a (6-4) PP photolyase or a CPD photolyase and exposed to opposite both the UV lesions, Rad18 depletion has a very severe photoreactivating light † ‡ − effect on the replication of lesion containing plasmid. Photolyase siRNA UV* PR Mutation frequency (×10 5) As we have shown previously, and determined again here for the effects of Polη and Polζ (Rev3) depletion on TLS opposite NC§ — + 13.2 ± 2.4 both the DNA lesions, the TLS frequency was reduced to ∼12% Rad18 — + 12.6 ± 3.1 in cells depleted for both the Pols (Table 1). TLS was reduced NC ++ 48.2 ± 4.6 even further in Rad18-depleted cells, as opposite both the Rad18 ++ 17.2 ± 3.6 lesions, TLS occurred at a frequency of only ∼5% (Table 1), (6-4)PP Polη ++ 104.6 ± 4.2 which presumably results from the residual Rad18 that remains Photolyase Polκ ++ 32.6 ± 2.8 in cells treated with Rad18 siRNA. We infer from these obser- Rev3 ++ 30.8 ± 3.6 vations that all of the TLS pathways are inhibited in the absence Polκ + Rev3 ++ 15.8 ± 2.7 of Rad18. Polη + Rad18 ++ 19.2 ± 3.8 Polκ + Rad18 ++ 15.6 ± 2.8 Requirement of Rad18 for UV Mutagenesis Resulting from TLS Rev3 + Rad18 ++ 16.1 ± 2.3 Opposite CPDs in the Chromosomal cII Gene in Mouse Cells. To NC — + 12.6 ± 2.2 provide further evidence that Rad18 plays an indispensable role Rad18 — + 11.4 ± 2.6 in TLS, we examined the effects of Rad18 depletion on UV CPD NC ++ 26.3 ± 3.2 mutagenesis in mouse cells. For this purpose, we separately de- Photolyase Rad18 ++ 13.6 ± 3.8 termined the effects of Rad18 depletion on UV mutagenesis Rev3 ++ 39.2 ± 4.3 resulting from TLS through CPDs and (6-4) photoproducts in Rev3 + Rad18 ++ 15.3 ± 3.1 the cII transgene that has been integrated into the genome in *5 J/m2 of UVC (254 nm) light. fi † a Big Blue mouse embryonic broblast (BBMEF) cell line (21). Photoreactivation with UVA (360 nm) light for 3 h. ‡ In a number of different experiments, this system has been Mutation frequencies were determined from averages of six to seven in- shown to exhibit similar mutational patterns as those observed dependent experiments. with endogenous chromosomal genes (21–23). §NC, negative control siRNA.

Yoon et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 The siRNA knockdown of Rad18 conferred a large reduction of Rad18 depletion on the mutational pattern resulting from − in mutation frequency from ∼48 × 10 5 in UV-irradiated control TLS opposite CPDs, we analyzed the types of mutations that − (NC) siRNA treated cells to ∼17 × 10 5 in Rad18-depleted cells were formed in the cII gene in UV-irradiated mouse cells (Table 2). Similar to the results we have reported previously (19), expressing the (6-4) photolyase and exposed to photoreactivating the siRNA knockdown of Polη results in a highly elevated fre- light. As we have shown previously (19), in control BBMEF cells − quency of ∼105 × 10 5, whereas the knockdown of either Polκ or treated with NC siRNA, a large majority of UV-induced muta- − Polζ confers a reduction in mutation frequencies to ∼30 × 10 5, tions resulting from TLS opposite CPDs are clustered at par- and the simultaneous knockdown of Polκ and Polζ reduces ticular dipyrimidine sites labeled with numbers 1–11 (Fig. 1A). − mutation frequencies to a level (∼16 × 10 5) similar to that seen Importantly, we ﬁnd that none of the hot spots characteristic of in unirradiated cells (Table 2). The indispensability of Rad18 for TLS opposite CPDs remain in UV-irradiated Rad18-depleted UV mutagenesis resulting from TLS opposite CPDs would pre- cells (Fig. 1A), and the pattern of mutations that occur becomes dict that the elevated mutagenesis in Polη-depleted cells will not similar to that in unirradiated cells treated with NC siRNA or occur in Rad18-depleted cells and that UV mutagenesis in with Rad18 siRNA (Fig. 1B). Thus, we conclude that Rad18 is Rad18-depleted cells will not be affected when either Polκ or essential for mutagenesis resulting from TLS opposite CPDs. Polζ have also been depleted. As shown in Table 2, UV induced mutation frequencies in Rad18-depleted cells remained the Requirement of Rad18 for UV Mutagenesis Resulting from TLS same, regardless of whether Polη, Polκ, or Polζ was depleted Opposite (6-4) Photoproducts in the Chromosomal cII Gene in along with Rad18, and in all cases, the frequency of UV-induced Mouse Cells. To examine the effects of Rad18 depletion on mu- mutations resulting from TLS opposite CPDs declined to levels tagenesis resulting from replicative bypass of (6-4) photo- similar to those in unirradiated cells. products formed at various dipyrimidine sites in the mouse cII UV-induced mutations occur predominantly by C-to-T tran- gene, CPDs from UV-irradiated cells were selectively removed sitions in human cells (23–25) and a similar mutational spectrum from the genome by expressing a CPD photolyase gene and ex- is observed in the mouse cII gene (21). To determine the effects posing cells to photoreactivating light (21). As shown in Table 2,

Fig. 1. Mutational spectra opposite CPDs in UV irradiated mouse (BBMEF) cells treated with Rad18 siRNA. Mutational spectrum was determined for the cII gene in cells exposed to 5 J/m2 of UV light; only the sequence for nucleotide positions 25–288 is shown because the UV-induced mutations are clustered in this region. (A) Mutations resulting from TLS opposite CPDs in UV irradiated cells treated with control (NC) siRNA (above the sequence) or treated with Rad18 siRNA (below the sequence). The (6-4) photoproducts were removed from the genome by expressing a (6-4) photolyase gene and exposing cells to photoreactivating light. The positions of mutational hot spots opposite CPDs (numbers 1–11) are indicated. (B) Mutations in non-UV–irradiated cell line expressing the (6-4) photolyase, and treated with control (NC) siRNA (above the sequence) or treated with Rad18 siRNA (below the sequence).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1204105109 Yoon et al. Downloaded by guest on September 27, 2021 in control siRNA-treated UV-irradiated mouse cells expressing Rad18 depleted cells, none of the UV induced mutational hot the CPD photolyase gene and exposed to photoreactivating light, spots remain (Fig. 2A), and the random pattern of mutations − mutation frequency increased to ∼26 × 10 5 compared with the becomes similar to that in unirradiated cells (Fig. 2B). From − spontaneous mutation frequency of ∼13 × 10 5 in unirradiated these observations, we conclude an essential role of Rad18 in cells. In Rad18-depleted cells, the frequency of UV-induced mutagenesis resulting from TLS opposite (6-4) photoproducts. mutations arising from replicative bypass of (6-4) photoprod- − ucts declined to ∼14 × 10 5, a level similar to that observed in Discussion unirradiated cells. Our observations indicate an essential role for Rad18 in TLS As we have shown previously, during replication in mouse and during replication in mouse and human cells. Briefly, we find human cells, TLS opposite (6-4) photoproducts occurs via a that: (i) The UV sensitivity of Rad18-depleted mouse and hu- predominantly error-free pathway dependent upon Polζ, and man cells is not enhanced upon the simultaneous depletion of by a more mutagenic pathway that requires Polη or Polι (20). any of the TLS Pols involved in UV lesion bypass. The epistasis Hence, in cells depleted of both Polη and Polι, mutagenic TLS of Rad18 with TLS Pols is in accord with a role for Rad18 in opposite (6-4) photoproducts is abrogated because, in the ab- promoting lesion bypass by TLS Pols. (ii) In Rad18-depleted sence of both these Pols, only the Polζ-controlled error-free human cells, replication of undamaged plasmid is not affected pathway operates. On the other hand, in the absence of Polζ, the but the replication efficiency of a plasmid carrying a cis-syn TT frequency of mutagenic TLS opposite (6-4) photoproducts is dimer or a (6-4) TT photoproduct is greatly reduced. (iii) Op- elevated because then TLS occurs via the Polη- and Polι-de- posite both the UV lesions, the TLS frequency is greatly reduced pendent error-prone pathways. As would be expected from the in cells depleted for Rad18, indicating that all of the TLS indispensability of Rad18 for mutagenic TLS opposite (6-4) pathways are inhibited. (iv) As determined from the analyses of photoproducts, simultaneous depletion of Rev3 with Rad18 led mutation frequencies and mutational spectra resulting from TLS to a drastic reduction in the elevated mutation frequencies that opposite CPDs and opposite (6-4) photoproducts in mouse cells, − occur upon the depletion of Rev3 alone (∼39 × 10 5), and mu- Rad18 is essential for mutagenic TLS opposite both these UV − tation frequencies declined to a level (∼15 × 10 5) similar to that induced DNA lesions. Because mutagenic TLS opposite CPDs observed for unirradiated cells treated with control (NC) siRNA depends upon Pols κ and ζ (19), and mutagenic TLS opposite − (∼13 × 10 5) (Table 2). (6-4) photoproducts requires Pols η and ι (20), Rad18 controls

As has been shown previously (20), mutations at (6-4) pho- TLS by all these Pols. GENETICS toproducts in the cII gene occur primarily at five dipyrimidine Our observations have provided clear evidence that Rad18 plays sites (numbers 1–5), and in the sequence data shown in Fig. 2A, a crucial role in TLS in mammalian cells. Because Rad18 asso- clustering of mutations is seen at four of these five hot spot sites, ciates with Rad6 and because the human Rad6-Rad18 complex numbers 1, 2, 4, and 5, but not at site number 3, which is the least monoubiquitylates PCNA at K164 (26), we presume that PCNA frequently mutated site among the five sites. Importantly, in ubiquitylation is a prerequisite for TLS in mammalian cells as

Fig. 2. Mutational spectra opposite (6-4) photoproducts in UV irradiated mouse (BBMEF) cells treated with Rad18 siRNA. (A) Mutations in the cII gene resulting from TLS opposite (6-4) photoproducts in UV-irradiated (5 J/m2) cells treated with control (NC) siRNA (above the sequence) or treated with Rad18 siRNA (below the sequence). CPDs were removed from the genome by expressing a CPD photolyase gene and by exposing cells to photoreactivating light. The positions of mutational hot spots opposite (6-4) photoproducts (numbers1–5) are indicated. (B) Mutations in the non-UV–irradiated cell line expressing the CPD photolyase, and treated with control (NC) siRNA (above the sequence) or treated with Rad18 siRNA (below the sequence).

Yoon et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 well. The observations with human cell-free extracts that repli- 2% of mutational events, and that opposite this UV lesion, Polζ cation through a cis-syn TT dimer could occur in the absence of functions in TLS in a nonmutagenic manner in both human and Rad18 as well as with the K164R mutant PCNA (13, 14) might mouse cells (20). In contrast, TLS opposite a (6-4) TT photo- reflect the possibility that the complexity of cellular mechanisms product carried in a gapped plasmid in mouse cells occurred in that operate when the replication fork stalls at the lesion site and a very highly mutagenic manner, such that ∼75% of TLS events TLS occurs is not recapitulated in these in vitro studies. were mutagenic, and Polζ was responsible for mediating this Our observations that Rad18 is required for UV-induced highly error-prone mode of TLS (27). Hence, we infer from mutations resulting from replication through CPDs and (6-4) these various observations that the results obtained from TLS photoproducts induced in the chromosomal cII gene in mouse analyses using a gapped plasmid are not informative for un- cells have indicated the indispensability of Rad18 for mutagenic derstanding how TLS operates during replication. TLS by Pols κ and ζ opposite CPDs, and for mutagenic TLS In conclusion, from analyses of replication of plasmids carry- opposite (6-4) photoproducts by Pols η and ι. Thus, Rad18 is ing a cis-syn TT dimer or a (6-4) TT photoproduct in human required for promoting the function of each of the Pols involved cells, and from determination of mutation frequencies and mu- in TLS opposite these DNA lesions. Furthermore, our observa- tational spectra formed opposite CPDs or (6-4) photoproducts in tion that replication through a cis-syn TT dimer or a (6-4) TT the chromosomal cII gene in mouse cells, we show that Rad18 is photoproduct carried on a duplex plasmid is severely curtailed in indispensable for TLS and damage-induced mutagenesis. This Rad18-depleted human cells, and that the frequency of TLS conclusion implies that the observations that have been reported opposite both the UV lesions is greatly reduced, have provided for the much less important role of Rad18 in lesion bypass in additional and direct evidence for the indispensability of Rad18 chicken DT40 cells (17) do not pertain to human or mouse cells. for lesion bypass during replication. These observations differ from the results that have been reported for TLS in a gapped Materials and Methods −/− plasmid carried in mouse Rad18 cells, because in that case, TLS was examined in human cells using SV40 origin based duplex plasmids, only the efficiency of TLS was affected but the relative pro- which carried a cis-syn TT dimer or a (6-4) TT photoproduct on the leading portion of error-free vs. mutagenic TLS remained the same as in strand DNA template. Frequencies of UV induced mutations that resulted wild-type cells (15), indicating that TLS via the various pathways specifically from replication through CPDs or (6-4) photoproducts formed at could still occur during gap repair in the absence of Rad18. The TT, TC, and CC dipyrimidine sites were examined in the cII gene carried in big indispensability of Rad18 for TLS during replication but not blue mouse embryonic fibroblast (BBMEF) cells. The details for construction for TLS in a gapped plasmid adds further evidence that the ge- of lesion containing plasmid and for lesion bypass assays in human cells and for mutational studies in mouse cells have been described previously (19, 20), netic control of TLS that occurs during replication differs in and the details that deal more specifically with Rad18 are provided in the many ways from that in gap repair. In this regard, in a previous SI Text. study, we provided evidence that TLS opposite a (6-4) TT photoproduct carried on the leading or the lagging strand DNA ACKNOWLEDGMENTS. This work was supported by National Institutes of template of a duplex plasmid in human cells incurs in less than Environmental Health Sciences Grant ES012411.

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