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 di- mer 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.