Requirement of Rad5 for DNA Polymerase Z-Dependent Translesion Synthesis in Saccharomyces Cerevisiae

Requirement of Rad5 for DNA Polymerase z-Dependent Translesion Synthesis in Saccharomyces cerevisiae

Vincent Page`s,* Anne Bresson,† Narottam Acharya,* Satya Prakash,* Robert P. Fuchs‡ and Louise Prakash*,1 *Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1061, †Department Inte´grite´ du Ge´nome, UMR7175 Centre National de la Recherche Scientiﬁque/Universite´ Louis Pasteur, Ecole Supe´rieure de Biotechnologie de Strasbourg, Bld S. Brant, BP 10413, 67412 Illkirch, France and ‡CNRS, Unite´ Propre de Recherche 3081, Genome Instability and Carcinogenesis Conventionne´ par l’Universite´ d’Aix-Marseille 2, 13402 Marseille Cedex 20, France Manuscript received May 5, 2008 Accepted for publication July 14, 2008

ABSTRACT In yeast, Rad6–Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases h or z or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities—a ubiquitin ligase that promotes Mms2–Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5’s ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polz. Using duplex plasmids carrying different site-speciﬁc DNA lesions—an abasic site, a cis–syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct—we show that Rad5 is needed for Polz-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.

NtheyeastSaccharomyces cerevisiae, the Rad6–Rad18 the nucleotide opposite the lesion site and another I ubiquitin-conjugating enzyme complex (Bailly et al. Pol carries out the subsequent extension reaction 1994, 1997) promotes replication through DNA lesions (Bresson and Fuchs 2002; Prakash et al. 2005). Polz, by DNA polymerase (Pol) h-andz-mediated translesion composed of the Rev3 catalytic and Rev7 accessory synthesis (TLS) (Nelson et al.1996b;Johnson et al. subunits (Nelson et al. 1996b), plays an important role 1999b; Prakash et al. 2005), and by a Rad5–Mms2– in TLS by extending from the nucleotide inserted Ubc13-dependent pathway in which the gaps that form opposite a DNA lesion by another Pol ( Johnson et al. opposite DNA lesion sites could be filled in by template 2000, 2001, 2003; Haracska et al. 2001; Prakash et al. switching (Torres-Ramos et al. 2002; Gangavarapu 2005; Nair et al. 2006, 2008). et al. 2006; Blastyak et al.2007).Polh is unique among In the Mms2–Ubc13–Rad5-dependent postreplication eukaryotic TLS Pols in its proficient and error-free ability repair (PRR) pathway, the Mms2–Ubc13 ubiquitin- to replicate through UV-induced cyclobutane pyrimi- conjugating enzyme complex in conjunction with Rad5 dine dimers (CPDs) ( Johnson et al. 1999b); hence carries out the lysine 63-linked polyubiquitination of inactivation of Polh in humans and deletion of the yeast PCNA at its K164 residue. In DNA damaged yeast cells, RAD30 gene, which encodes Polh, leads to a high PCNA is first monoubiquitinated at the K164 residue by incidence of UV mutagenesis (McDonald et al. 1997; Yu Rad6–Rad18 and subsequently, this lysine residue is poly- et al. 2001; Stary et al. 2003) and in humans causes the ubiquitinated via the action of the Mms2–Ubc13–Rad5 cancer-prone syndrome, the variant form of xeroderma complex (Hoege et al.2002).Rad5,amemberofthe pigmentosum ( Johnson et al. 1999a; Masutani et al. SWI/SNF family of ATPases ( Johnson et al. 1992, 1994), 1999). Although proficient replication through a DNA exhibits a DNA helicase activity that is highly specialized lesion such as a CPD or an 8-oxoguanine can be for promoting replication fork regression (Blastyak mediated by a single TLS Pol, as for example, by Polh et al. 2007). Rad5 additionally harbors a C3HC4 motif (Johnson et al. 1999b; Haracska et al. 2000), replication characteristic of ubiquitin ligases (Zachariae et al. 1998; through many DNA lesions requires the consecutive Joazeiro et al. 1999; Lorick et al.1999;Fang et al. 2000). action of two different Pols, in which one Pol inserts In yeast cells, Rad5 physically associates with the Mms2– Ubc13 complex via Ubc13, and this association requires the C3HC4 motif of Rad5; Rad5 also interacts with the 1Corresponding author: Department of Biochemistry and Molecular lrich entsch Biology, University of Texas Medical Branch, 301 University Blvd., Rad6–Rad18 complex (U and J 2000). Galveston, TX 77555-1061. E-mail: [email protected] Mutational inactivation of the DNA helicase activity or

Genetics 180: 73–82 (September 2008) 74 V. Page`s et al. the ubiquitin ligase activity of Rad5 causes the same high also carried on a duplex plasmid. Also in this plasmid, degree of defectiveness in the repair of discontinuities replication initiates from a single-origin site and pro- that form in the newly synthesized strand in UV-damaged ceeds through a site-specific DNA lesion (Baynton et al. cells as that in the rad5D mutant, indicating that both 1998; Bresson and Fuchs 2002). From all these studies these activities are important for Rad5 function in PRR we conclude a requirement of Rad5 for TLS dependent (Gangavarapu et al. 2006). The direct involvement of upon Polz. We elaborate upon the possible implications Rad5 DNA helicase activity in promoting template of these observations for Rad5’s role in TLS opposite a switching and thereby enabling the use of the nascent diverse array of DNA lesions. lagging strand as the template for synthesizing DNA complementary to the damaged region provides for an MATERIALS AND METHODS important means by which replication through the lesion site can be accomplished in an error-free manner Yeast strains: For the study of AP bypass (Tables 1 and 2), we (Blastyak et al. 2007). used strain EMY74.7 (MATa his3D-1 leu2-3,-112 trp1D ura3-52) As expected from the roles of Rad5 and Polh in from which the two AP endonuclease genes APN1 and APN2 have been deleted to prevent the repair of the abasic site. promoting error-free synthesis through UV lesions, the Additionally, we deleted the MSH2 gene to prevent the frequency of UV-induced forward mutations at the removal of the mismatch loop present opposite the AP site CAN1 locus is greatly enhanced in the rad5D rad30D dou- (Figure 1A). We refer to this apn1D apn2D msh2D strain as the ble mutant compared to that in either single mutant wild-type strain since it is wild type with respect to the proteins (Johnson et al. 1999c). Curiously, however, the loss involved in lesion bypass. The various genomic deletion and other mutations were introduced into this apn1D apn2D msh2D of Rad5 function adversely affects UV-induced rever- strain (Pages et al. 2008). For the study of AAF and UV lesion sion of ochre alleles (Lemontt 1971; Lawrence and bypass, plasmids containing a single lesion (Baynton et al. Christensen 1978; Johnson et al. 1992). For example, 1998; Bresson and Fuchs 2002) were transformed into the UV-induced reversion of arg4-17 to Arg1 is reduced yeast strain EMY74.7 or into its rad30D, rad5D,orrad30D rad5D 10-fold in the rad5D strain ( Johnson et al. 1992). derivatives (Tables 3–6). To study the requirement of Rad5 in TLS opposite an AP site Sequence analyses of UV-induced arg4-17 to ARG41 (Table 1), we used a rad5D strain in which the wild type or its revertants has indicated the reversion to be predomi- mutant derivatives are carried on a YCplac133-based plasmid, nantly a T / C transition of T127 that would constitute and which contains the ARS1 origin of replication, the cen- the 39T of a potential TT photoproduct (Zhang and tromeric CEN4 region, and the LEU2 gene. The plasmid, pR5- Siede 2002). To delineate whether the function of Rad5 28, expresses the wild-type Rad5 protein (RAD51). Plasmid pR5-30 carries the mutations D681, E682 / AA in RAD5,which in UV-induced mutations at arg4-17 was mediated in inactivate the ATPase and DNA helicase activities of Rad5 (rad5- collaboration with Mms2–Ubc13, in a previous study, ATPase mutant), and plasmid pR5-19 carries the mutations / we examined the incidence of UV-induced reversion C914, C917 AA in the C3HC4 ring-finger motif that abolishes of arg4-17 in the mms2D, ubc13D, and rad5D strains the ubiquitin ligase function (rad5-Ub ligase mutant). (Gangavarapu et al. 2006). However, we found that in Duplex plasmids: Double-stranded, closed circular plasmids were generated using the gapped-duplex method (Broschard this role, Rad5 functioned independently of Mms2– et al. 1999). For the AP-containing plasmid, the damaged strand Ubc13. Moreover, the inactivation of Rad5 helicase carries the TRP1 gene that allows for the selection of trans- function or of its ubiquitin ligase function had no formants resulting from the replication of this strand. The AP site adverse affect upon UV mutagenesis. From such obser- is located in a heteroduplex leader sequence that we inserted in vations, we concluded a structural role of Rad5 in UV the URA3 gene at its 59 end in two different orientations, which thereby presents the AP site on the leading or the lagging DNA mutagenesis at sites such as arg4-17. However, from all strand during replication (Figure 1A). This construct was the previous studies, only a very limited role of Rad5 in obtained by the ligation of a 16-mer oligonucleotide 59-GGAAG UV mutagenesis that is restricted to the reversion of CAATXGTACGG-39 (where X denotes a tetrahydrofurane- ochre alleles could be inferred. type AP site) into a gapped-duplex structure. This leader Recently, we have reported on our analyses of TLS sequence where the damaged oligonucleotide is ligated is in frame with the URA3 gene, whereas the opposite strand opposite an abasic (AP) site in yeast cells wherein we contains a 11 frameshift that inactivates the ura3 gene. Hence employed a plasmid system in which bidirectional cells arising from the replication through the AP site by TLS replication proceeds from a yeast origin of replication are Ura1, and cells that underwent non-TLS-mediated repli- (Pages et al. 2008). We showed that the rate and genetic cation of the damaged strand (such as copy-choice events) are control of TLS for both the leading and lagging DNA UraÀ. All cells resulting from the replication of the damaged strand are Trp1 (Figure 1A). Plasmids containing a single G- strands is very similar and that Polz and PCNA ubiquiti- AAF adduct, a cis–syn TT dimer, or a (6-4) TT photoproduct nation are indispensable for TLS on both the DNA (Figure 1B) were constructed as in (Baynton et al. 1998; strands (Pages et al. 2008). Using this plasmid system, Bresson and Fuchs 2002). These constructs contain a short we have now examined the effects of the rad5D, mms2D, sequence heterology opposite the lesion site to be able to and ubc13D mutations on TLS through the AP site. In monitor TLS (Figure 1C). Yeast transformation and identification of TLS products: addition, we have carried out studies of TLS through a Plasmids carrying the AP site were introduced into yeast cells number of other DNA lesions—a cis–syn TT dimer, a by electroporation as previously described (Pages et al. 2008). (6-4) TT photoproduct, and an AAF adduct—that are Briefly, cells were grown to exponential phase in YPD, washed Role of Yeast Rad5 in TLS 75 several times, and concentrated in 1 m sorbitol. Then 20 ng of AP site are Ura1. The frequency of TLS among the plasmid DNA were electroporated, and 1 ml of YPD was added. transformants is determined from the ratio of colonies After incubation for 40 min at 30° the cell suspension was that grow on –trp –ura media vs. those that grow on –trp washed with water before plating on selective media: either synthetic complete media lacking tryptophan (SC Àtrp) to (Figure 1A). select for the cells that had replicated the damaged strand or As shown in Table 1, TLS accounts for 5–6% of AP SC Àtrp lacking uracil (SC Àtrp Àura) to select for plasmids bypass in wild-type cells, and the frequency of TLS is that underwent TLS through the AP site. The ratio of Ura1/ reduced by 90% in rev3D cells. Interestingly, the 1 Trp colonies indicated the TLS frequency. To identify the frequency of TLS also shows a large reduction in the nucleotide inserted opposite the AP site during TLS, Ura1 colonies were analyzed by direct PCR followed by digestion by rad5D strain nearly similar to that in the rev3D strain. a restriction enzyme (Pages et al. 2008). TLS events opposite To determine whether the Rad5 helicase and the ubiq- the cis–syn TT dimer, (6-4) TT photoproduct, and the G-AAF uitin ligase activities contribute to TLS opposite the AP adduct were detected by colony hybridization using strand- site, we introduced into rad5D cells a plasmid carrying resson uchs specific oligonucleotides (B and F 2002). The the wild-type RAD5 gene or the rad5 mutant gene in- frequency of mutagenic TLS through the G-AAF adduct was determined by overlaying SC Àtrp plates with X-Gal- activated for the helicase or the ubiquitin ligase func- containing agarose (Bresson and Fuchs 2002). The 11 and tion. We found that both the helicase-defective and the À1 frameshift mutations in the GTTT and GCCC context, ubiquitin-ligase-defective rad5 mutant genes restored respectively, restore the lacZ gene reading frame. The molec- TLS in rad5D cells to almost the same level as that con- ular nature of the induced mutation was further confirmed by ferred upon by the wild-type RAD5 gene. Hence both sequencing. Physical interaction of Rad5 with Rev1 and Polz: For the Rad5 helicase and ubiquitin ligase activities are physical interaction studies, RAD5, REV1, and REV3 genes dispensable for Rad5’s role in mediating TLS opposite were inserted into the vector pBJ842 to produce an amino- an AP site. In keeping with the lack of requirement of terminal glutathione-S-transferase (GST) fusion protein. To the ubiquitin ligase activity of Rad5, we find that the z purify Pol , GST–REV3 protein was coexpressed with Rev7 in mms2D or ubc13D mutations also cause no significant yeast strain BJ5464. Proteins were purified on glutathione- sepharose beads by using a protocol described earlier ( Johnson impairment of TLS opposite this lesion site (Table 1). et al. 2006). To obtain untagged proteins, GST fusion proteins Sequence analysis of TLS products from wild-type bound to glutathione-sepharose beads were treated overnight at cells has shown that opposite an AP site on both DNA 4° with PreScission protease which cleaved between the GST tag strands an A is incorporated with a frequency of 70%, a and the protein of interest. The physical interaction of Rad5 C is incorporated with a frequency of 25%, and G and with Rev1 and Polz was examined using a protocol similar to that described earlier (Acharya et al. 2005). Briefly, GST–Rad5 or T insertions are much rarer. As shown in Table 2, the GST alone was incubated with Rev1, and GST–Polz was in- predominance of A insertion persists in the rev3D strain cubated with Rad5 in buffer I (50 mm Tris-HCl, pH 7.5, 150 mm and also in the rad5D and mms2D strains. Thus, even NaCl, 5mm dithiotheritol, 0.01% NP-40, and 10% glycerol) in a though the frequency of TLS is greatly reduced in the m 20 lreactionat4° for 30 min, followed by 10 min at 25°.Tothis rev3D and rad5D strains, the nucleotide insertion pat- mixture, 20 ml glutathione-sepharose beads were added and further incubated for 1 hr with constant rocking at 4°. The beads tern remains about the same in these mutant strains as were spun down and the unbound protein was collected. in the wild-type strain. Further, the beads were washed thoroughly three times with Rad5 is not required for error-free TLS opposite a 10 vol of buffer I. Finally, the bound proteins were eluted with 20 cis–syn TT dimer by Polh: The duplex plasmid system m l of SDS loading buffer. Various fractions were resolved on a used for determining the role of Rad5 in promoting 12% denaturing polyacrylamide gel, followed by Coomassie blue R-250 staining. TLS through a cis–syn TT dimer, a (6-4) TT photoproduct, and an AAF adduct, is shown in Figure 1B, and the sequences resulting from error-free and mutagenic TLS events are shown in Figure 1C. In wild-type yeast cells RESULTS TLS accounts for 13% of bypass through the TT dimer Requirement of Rad5 for TLS opposite an AP site: and almost all of it is error free (Table 3). TLS is reduced The details of the plasmid system that was used for these by 50% in the rad30D strain and about the same level studies have been described previously (Pages et al. of reduction occurs in the rad5D strain. Interestingly 2008). Briefly, the plasmid carries a yeast replication and importantly, a synergistic decline in TLS frequency origin ARS1 and a site-specific AP site located on the occurs in the rad30D rad5D strain such that the level leading or the lagging DNA strand. Since a tetrahydro- of TLS decreases by .15-fold (Table 3). From these furan lesion was used as an AP site analog, there may observations we infer a role for Rad5 in promoting TLS exist differences in its bypass from that of an AP site. In through the TT dimer via a pathway that acts indepen- this system, replication of the AP site-containing DNA dently of Polh. strand, regardless of whether AP bypass occurred by TLS Requirement of Rad5 for TLS opposite a (6-4) TT or by any other mechanism, such as template switching, photoproduct: In wild-type yeast cells, TLS accounts for results in a Trp1 cell. The AP site is present in a 4% of bypass opposite the (6-4) TT photoproduct, of heteroduplex leader sequence in the URA3 gene and which 1.6% results from error-free synthesis through cells harboring plasmid resulting from TLS through the the photoproduct and 2.5% results from mutagenic 76 V. Page`s et al.

Figure 1.—Plasmids used for TLS assays. (A) Plasmid used for TLS opposite an AP site. The plasmid carries an AP site in the leading or the lagging DNA strand. TLS through the AP site results in Ura1 cells and the frequency of Ura1 cells among Trp1 cells reflects the TLS frequency. (B) Plasmid used for TLS opposite a cis–syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct. In this plasmid, the DNA lesion is contained within a heteroduplex sequence (see C) which allows for the detection of TLS events as follows. For each strain, individual colonies are probed with 32P-labeled oligonucleotides that specifically hybridize with either the lesion-containing target strand or the marker strand. Colonies that hybridize with the target strand are scored as TLS events whereas colonies that hybridize with the marker strand could reflect lesion bypass by a damage avoidance (DA) pathway. For the UV photoproducts, the molecular nature of the TLS event (i.e., error free vs. mutagenic) is determined following isolation of the plasmid DNA from TLS-positive yeast colonies, transformation into Escherichia coli, and sequencing. For the G-AAF adduct, the total extent of TLS is determined by colony hybridization as described above, while mutagenic TLS is scored directly in yeast by overlaying the transformation plates with X-Gal-containing agarose (Bresson and Fuchs 2002). Indeed, the frameshift mutations induced by the G-AAF adduct (11 and À1 in the GTTT and GCCC context, respectively) restore the lacZ gene reading frame and appear as blue yeast colonies. (C) The heteroduplex region containing the DNA lesion and the sequence changes resulting from the error-free and mutagenic TLS events through the lesions carried on the plasmid shown in B. bypass that involves a 39T / C transition (Bresson and of error-free TLS is not affected (Bresson and Fuchs Fuchs 2002) (Table 4, Figure 1C). In the rad30D strain, 2002). Similar to that in rad30D, the incidence of mutagenic TLS is reduced by .10-fold while the level mutagenic TLS shows a large decrease in the rad5D Role of Yeast Rad5 in TLS 77

TABLE 1 bypass events, and a great majority of the events are TLS frequencies opposite an AP site in different yeast strains error free (6.5%), resulting from a C insertion opposite the G-AAF adduct, whereas a small proportion of TLS DNA % TLS events (0.2%) result from frameshifting of the primer Strain strand Trp1 Ura1 TLS (% of WT)a strand which generates a 11 T insertion (Table 5, Figure 1C). In the rad30D strain, the frequencies of both error- WTb Leading 3090 182 5.9 100 Lagging 6460 304 4.7 100 free TLS and mutagenic TLS decrease 4-fold. Impor- Total 9550 486 5.1 100 tantly, we ﬁnd that in the rad5D strain, the incidence of error-free TLS declines by .30-fold, from 6.5% in the rev3Db Leading 11580 58 0.5 8.5 wild-type strain to 0.2% in the rad5D strain, and there is Lagging 26160 90 0.3 7.3 Total 37740 148 0.4 7.7 almost a complete absence of mutagenic TLS (Table 5). Furthermore, the magnitude of decline in the in- D rad5 Leading 10620 113 1.1 18.1 cidence of error-free and mutagenic TLS in the rad5D Lagging 25630 168 0.7 13.9 D D Total 36250 281 0.8 15.2 rad30 strain resembles that in the rad5 mutant alone (Table 5), which implicates epistasis of Rad5 over rad5D with Leading 6590 385 5.8 99.2 Polh function in mediating TLS opposite the G-AAF RAD51 Lagging 11400 609 5.3 113.5 adduct. Total 17990 994 5.5 108.6 In the 39-GCCC context in the wild-type strain, error- rad5 ATPase Leading 3880 135 3.5 59.1 free TLS accounts for almost 99% of TLS, the remain- mutant Lagging 8430 409 4.9 103.1 der (1%) being mutagenic events resulting from the Total 12310 544 4.4 86.8 frameshifting of the template strand that generates a À1 rad5 Ub ligase Leading 9520 485 5.1 86.5 C replication product (Figure 1C). In the rad30D strain, mutant Lagging 24830 1205 4.9 103.1 the frequency of error-free TLS drops 10-fold and Total 34350 1690 4.9 96.7 mutagenic TLS is almost abolished, whereas in the mms2D Leading 1370 75 5.5 92.9 rad5D and the rad5D rad30D strains, no TLS products Lagging 2660 92 3.5 73.5 were recovered (Table 6). Thus, in both sequence con- Total 4030 167 4.1 81.4 texts, TLS opposite the G-AAF adduct is affected to a much ubc13D Leading 420 41 9.8 165.7 greater degree in the rad5D strain than in the rad30D Lagging 2470 118 4.8 101.5 strain, and Rad5 displays epistasis over Polh action. Total 2890 159 5.5 108.1 Physical interaction of Rad5 with Rev1: As we WT, wild type. elaborate in the discussion, our genetic observations a The frequency of TLS in the WT strain for each of the indicating a requirement of Rad5 for TLS opposite DNA DNA strands and for the total of both DNA strands is set at lesions such as an AP site, a (6-4) TT photoproduct, and 100%. b a G-AAF adduct, support a role for Rad5 in Polz- The data for WT and rev3D strains are from Pages et al. (2008). dependent TLS. Since Rad5 functions in this role as a structural element (see discussion), we examined the possibility of whether Rad5 is involved in direct physical strain, but unlike that for rad30D, the frequency of error- interactions with Polz, or with Rev1, which is a necessary free TLS is also reduced 4-fold in the rad5D strain element for Polz function in TLS. We have shown (Table 4). Thus, whereas the absence of Polh affects only previously that Rev1 forms a physical complex with Polz the mutagenic component of TLS, the absence of Rad5 and have suggested a role for Rev1 in the targeting of affects the incidence of both the mutagenic and error- Polz to the replication fork stalled at a DNA lesion free modes of TLS. Moreover, and interestingly, the (Acharya et al. 2006). absence of Rad5 alone has the same adverse effect on To check for the physical interaction of Rad5 with TLS as the absence of both Rad5 and Polh, implicating Rev1 and with Polz, we bound a mixture of puriﬁed GST– an epistatic relationship of Rad5 with Polh function. Rad5 and Rev1 protein to the glutathione-sepharose Requirement of Rad5 for TLS opposite a guanine- beads, rocked it for 1 hr, followed by extensive washings AAF adduct: AAF predominantly forms an adduct at the with 150 mm NaCl-containing buffer before eluting it C8 position of guanine (Kriek et al. 1967). Previously with SDS-containing buffer. In such a system, GST fu- Bresson and Fuchs (2002) examined the genetic sion protein will bind to the beads and the interacting control of TLS through this adduct in two different protein will be pulled down only if it forms a stable sequence contexts, a 39-GTTT sequence, in which the complex. As shown in Figure 2, Rev1 eluted together adducted G is followed by 3 T’s on the 59 side in the tem- with Rad5, indicating that Rad5 forms a stable physical plate strand and a 39-GCCC sequence where the ad- complex with Rev1 at physiological salt concentration ducted G is followed by 3 C’s on the 59 side (Figure 1C). (Figure 2, lane 4). In the control experiments, Rev1 In a wild-type yeast strain, when the adduct is located in did not show any interaction with GST protein alone the 39-GTTT sequence, TLS accounts for 6.7% of (Figure 2, lane 8). We found no evidence for the inter- 78 V. Page`s et al.

TABLE 2 Types and frequencies of nucleotides incorporated opposite an AP site in the rev3D, rad5D, and mms2D strains

No. nucleotides inserted in TLS (%) Strain DNA strand No. A C G T WTa Leading 64 46 (71.9) 13 (20.3) 4 (6.3) 1 (1.6) Lagging 66 40 (60.6) 19 (28.8) 6 (9.1) 1 (1.5) Total 130 86 (66.2) 32 (24.6) 10 (7.7) 2 (1.5) rev3Da Leading 21 18 (85.7) 3 (14.3) 0 (0.0) 0 (0.0) Lagging 33 19 (57.6) 14 (42.4) 0 (0.0) 0 (0.0) Total 54 37 (68.5) 17 (31.5) 0 (0.0) 0 (0.0) rad5D Leading 32 26 (81.3) 5 (15.6) 0 (0.0) 1 (3.1) Lagging 32 27 (84.4) 5 (15.6) 0 (0.0) 0 (0.0) Total 64 53 (82.8) 10 (15.6) 0 (0.0) 1 (1.6) mms2D Leading 27 23 (85.2) 3 (11.1) 1 (3.7) 0 (0.0) Lagging 28 18 (64.3) 6 (21.4) 4 (14.3) 0 (0.0) Total 55 41 (74.5) 9 (16.4) 5 (9.1) 0 (0.0) WT, wild type. a The data for WT and rev3D strains are from Pages et al. (2008). action of Rad5 with Polz, as indicated from the absence shown that Polz is highly inefficient at inserting a of any Rad5 in the eluate (Figure 2, lane 12). The ability nucleotide opposite the AP site but it can proficiently of Rev1 to directly bind Rad5 may provide a means extend from the nucleotide inserted opposite the lesion whereby Rev1 targets Polz to the replication fork stalled site by another DNA Pol (Haracska et al. 2001). Since an at the DNA lesion site (see discussion). A is the most frequent nucleotide inserted opposite the AP site in our plasmid system, and because the replicative Pols such as Pold and Pole are able to insert an A DISCUSSION opposite this lesion, we have previously suggested that Our analyses of TLS opposite a number of site-specific following the A insertion by the replicative Pol, Polz DNA lesions carried on duplex plasmids have provided performs the extension reaction (Haracska et al. 2001). strong evidence for the requirement of Rad5 in TLS. We Since Rev1 promotes C insertion opposite the AP site discuss below the implications of these observations for (Nelson et al. 1996a; Haracska et al. 2002), which Rad5 involvement in Polz-dependent TLS and consider constitutes the second most frequent event (Pages et al. the possible ways by which Rad5 may act in such a role. 2008), that would also be followed by extension by Polz. Requirement of Rad5 for TLS mediated by Polz: AP Overall, because of the possible involvement of multiple site: Here we show that the rad5D mutation confers Pols at the insertion step, but of only Polz at the exten- almost the same high level of defect in TLS opposite an sion step, the requirement of Polz for AP bypass would AP site as does the rev3D mutation. Previously, we have be much more absolute than that of other Pols. Our

TABLE 3 TABLE 4 Effects of Polh and Rad5 on error-free and mutagenic TLS Effects of Polh and Rad5 on error-free and mutagenic TLS opposite a cis–syn TT dimer opposite a (6-4) TT photoproduct

TLS % TLS % Error Mutagenic Error Mutagenic Strain Total free (39T / C) Strain Total free (39T / C) WT 13.1 13.0 0.06 WT 4.1 1.6 2.5 (210/1604) (209/1604) (1/1604) (43/1042) (17/1042) (26/1042) rad30D 6.3 6.2 0.1 rad30D 2.0 1.8 0.18 (68/1084) (67/1084) (1/1084) (23/1104) (21/1104) (2/1104) rad5D 5.9 5.8 0.07 rad5D 0.47 0.47 #0.09 (85/1441) (84/1441) (1/1441) (5/1074) (5/1074) (0/1074) rad30D rad5D 0.8 0.8 #0.14 rad30D rad5D 0.53 0.53 #0.07 (6/703) (6/703) (0/704) (8/1507) (8/1507) (0/1507) WT, wild type. WT, wild type. Role of Yeast Rad5 in TLS 79

TABLE 5 Effects of Polh and Rad5 on TLS through a G-AAF adduct present in a 39-GTTT sequence

TLS % Mutagenic Strain Total Error free (11 fs) WT 6.7 6.5 0.16 (77/1151) (75/1151) (98/60320) rad30D 1.6 1.6 0.04 Figure 2.—Rad5 directly binds to Rev1 but not to Polz. (18/1143) (18/1143) (23/51307) Yeast Rev1 was mixed and incubated with GST–Rad5 (lanes rad5D 0.2 0.2 0.004 1–4) or with GST protein alone (lanes 5–8), and Rad5 was (1/576) (1/576) (1/21225) mixed and incubated with GST–Polz (Rev3–Rev7) (lanes 9– rad30D rad5D 0.2 0.2 ,0.01 12). One microgram of each protein was used in this study. (1/576) (1/576) (0/8982) After incubation, samples were bound to glutathione-sepharose beads for 1 hr, followed by multiple washings with buffer I fs, frameshift; WT, wild type. containing 150 mm NaCl and elution of the bound proteins with SDS-sample buffer. Aliquots of each sample before addi- observation that Rad5 is also required for TLS opposite tion to the beads (L), the flow through fraction (F), last washing z fraction (W), and the eluted proteins (E) were analyzed on an the AP site would imply that Rad5 is important for Pol ’s SDS-12% polyacrylamide gel developed with Coomassie blue. ability to function in TLS opposite this lesion site. (6-4) TT photoproduct: TLS opposite this photoproduct indispensability of Polz for both the error-free and either can occur in an error-free way by the insertion of mutagenic modes of TLS opposite the lesion site would an A opposite the 39T of the photoproduct by a DNA Pol then result from its absolute requirement at the exten- whose identity remains to be determined or can occur in sion step. Our finding that the level of both error-free a mutagenic way by the insertion of a G by Polh opposite and mutagenic TLS opposite this lesion site is greatly this lesion site ( Johnson et al. 2001; Bresson and Fuchs reduced in the rad5D strain adds further support for the 2002). Since Polz is highly inefficient at inserting a requirement of Rad5 in TLS mediated by Polz. nucleotide opposite the 39T of this lesion, but can carry G-AAF adduct: Previously we have shown that in yeast out efficient extension from the nucleotide inserted cells, TLS opposite this adduct absolutely requires Polz opposite this site by another Pol, we have previously (Baynton et al. 1998). Furthermore, Polh makes a very suggested that following nucleotide insertion opposite significant contribution to both the error-free and the 39Tsite by another Pol, Polz performs the subsequent mutagenic modes of TLS opposite this adduct; conse- extension reaction ( Johnson et al. 2000, 2001). A role for quently, a large reduction in both these modes of TLS Polh in promoting mutagenic TLS in which a 39T-to-C occurs in the rad30D strain (Bresson and Fuchs 2002). change occurs is in accordance with the ability of this Pol Here we show that TLS opposite the G-AAF adduct to insert a G opposite this lesion site ( Johnson et al. 2001; carried in both the sequence contexts is almost abol- Bresson and Fuchs 2002). Since the level of TLS ished in the rad5D strain. We interpret these various opposite a (6-4) TT photoproduct in yeast cells is greatly observations to suggest that TLS opposite the G-AAF reduced in the rev3D strain (Gibbs et al. 2005), the adduct occurs by nucleotide insertion opposite the lesion by Polh or by another Pol followed by extension TABLE 6 by Polz. The requirement of Polz as well as of Rad5 for Effects of Polh and Rad5 on TLS through a G-AAF adduct TLS opposite this lesion site reinforces further the need present in a 39-GCCC sequence for Rad5 in modulating Polz-dependent TLS. Rad5 is not required for Polh-mediated TLS TLS % opposite a cis–syn TT dimer: Although our observations Mutagenic with the above-noted lesions have provided clear evi- Strain Total Error free (À1 fs) dence that Rad5 can be as indispensable for TLS across these lesions as is Polz, they do not exclude the possible WT 7.0 6.9 0.028 (79/1129) (78/1129) (9/31448) requirement of Rad5 for Polh’s ability to carry out its rad30D 0.8 0.8 0.004 role in TLS opposite the (6-4) TT photoproduct or the (9/1134) (9/1134) (1/26952) G-AAF adduct. That is because opposite both these rad5D#0.2 #0.2 #0.006 lesions, the effect of the rad5D mutation is much more (0/576) (0/576) (0/17100) drastic than that of the rad30D mutation and the action rad30D rad5D#0.2 #0.2 ,0.004 of Polh is subserved under that of Rad5. Hence Rad5 (0/576) (0/576) (0/27814) could be indispensable not only for the action of Polz in fs, frameshift; WT, wild type. TLS but also for that of Polh. 80 V. Page`s et al.

Our analyses of TLS opposite a TT dimer, however, irradiated cells. In fact, in UV-damaged yeast cells, UV- clearly point to the lack of involvement of Rad5 in induced forward mutations at the CAN1s locus arise in modulating Polh action opposite this lesion site. Oppo- rad5D cells and their frequency is further enhanced in site a cis–syn TT dimer, Polh would function indepen- the rad5D rad30D strain (Johnson et al. 1992, 1999c). dently of Polz. Our finding that a synergistic decline in These observations have previously been ascribed to a error-free bypass occurs in the absence of both Polh and much greater dependence upon the error-prone Polz for Rad5 implies that Polh and Rad5 function indepen- lesion bypass when both the error-free pathways—Rad5- dently in promoting TLS opposite a TT dimer. Further- dependent template switching and Polh-dependent more, since Polz provides the only other TLS pathway in error-free TLS through the CPDs—have been inacti- addition to Polh, a role of Rad5 alternate to Polh implies vated. These seemingly disparate observations for the that Rad5 functions in modulating the Polz-dependent possible requirement of Rad5 in Polz-dependent TLS error-free bypass through this lesion site where, follow- when the lesion is carried on a duplex plasmid but the ing the insertion of an A opposite the 39T of the dimer cells have not been treated with a DNA damaging agent by a Pol other than by Polh, the subsequent extension vs. those carried out in UV-irradiated cells could best be reaction would be carried out by Polz. reconciled if we assume that in UV-damaged cells, an- Structural role of Rad5 in the assembly of Polz at the other protein is able to substitute for Rad5. Presumably, stalled replication fork: As determined from the analy- this other protein is nonfunctional in cells not treated ses of TLS opposite an AP site, we find that the with a DNA damaging agent because either it is not ex- inactivation of Rad5 helicase activity or of its ubiquitin pressed or it needs to be activated by a post-translational ligase activity has no significant effect on TLS, and the modification such as phosphorylation, and that occurs absenceofeitherMms2orUbc13alsoconfersno only when the cells have sustained significant levels of perceptible impairment of TLS. Since neither the DNA DNA damage. However, the requirement of Rad5 for UV helicase nor the ubiquitin ligase activities contribute to mutagenesis of certain ochre alleles such as arg4-17 Rad5’s role in TLS we surmise that Rad5 functions as a raises the possibility that even though the substitute pro- structural element in modulating Polz function in TLS. tein can function in lieu of Rad5 in most of the sequence Previous studies have indicated a role for Rad5 in contexts, the function of Rad5 is still required for promoting error-free postreplication repair by template modulating Polz-dependent TLS through some se- switching where its ubiquitin ligase activity is used for quence regions. PCNA polyubiquitination and its DNA helicase activity Although in its requirement for Rad5, Polz-dependent is used for fork regression. Here we suggest a third TLS through site-specific DNA lesions carried on duplex function for Rad5 wherein as a structural element, it plasmids in undamaged yeast cells differs from TLS in would modulate Polz’s function in TLS. How might UV-damaged cells where Rad5 can be dispensable for Rad5 contribute to Polz’s role in TLS? Previously we UV mutagenesis, TLS in both these cases depends upon have provided evidence for the formation of a complex PCNA ubiquitination (Stelter and Ulrich 2003; between Rev1 and Polz and have shown that this Haracska et al. 2004; Pages et al. 2008). The require- complex formation is important for Polz’s function in ment of PCNA ubiquitination for TLS opposite a single TLS (Acharya et al. 2006). Our observation that Rad5 DNA lesion in undamaged yeast cells as inferred from directly binds to Rev1 but not to Polz raises the pos- plasmid studies has suggested that the stalling of rep- sibility that physical association with Rad5 affects the licative DNA polymerase at the DNA lesion is sufficient ability of Rev1 to target Polz to the replication fork to generate a signal for Rad6–Rad18-mediated PCNA stalled at a DNA lesion. Two-hybrid analyses and co- ubiquitination and that a certain threshold level of DNA immunoprecipitation studies have shown that Rad5 damage is not needed for PCNA ubiquitination to occur exists in a complex with Rad6–Rad18 in yeast cells and (Pages et al. 2008). In recent biochemical experiments Rad5 binds this complex via Rad18 (Ulrich and in which processively moving yeast Pold was stalled in the Jentsch 2000). It is quite likely that the Rad6–Rad18– presence of PCNA or monoubiquitinated PCNA by nu- Rad5 complex binds to the template strand at the lesion cleotide omission, exchange with Polh could occur only site and from there it effects PCNA ubiquitination; in the presence of ubiquitinated PCNA and not with additionally, this complex could also have a role in unmodified PCNA (Zhuang et al. 2008). Hence, the promoting the assembly of the TLS Pols at the lesion available genetic and biochemical evidence supports the site. In that case, the binding of Rad5 by Rev1 could be view that PCNA ubiquitination provides a key mecha- important for the targeting of Polz to the DNA lesion nism for polymerase exchange to occur when the site where replication has stalled. replicative polymerase stalls at a DNA lesion or from Other considerations: The requirement of Rad5 for some other cause. Polz-dependent TLS that we have uncovered here from In contrast to the requirement of Rad6–Rad18- the studies done with duplex plasmids carrying a site- dependent PCNA ubiquitination for TLS as inferred specific DNA lesion stands in contrast to the lack of from plasmid studies and from the studies of DNA requirement of Rad5 in Polz-mediated TLS in UV- damage-induced mutagenesis, genetic analyses of Polz- Role of Yeast Rad5 in TLS 81 dependent spontaneous mutagenesis in yeast have in- Haracska, L., S.-L. Yu,R.E.Johnson,L.Prakash and S. Prakash, dicated that it can occur via two separate pathways—one 2000 Efficient and accurate replication in the presence of 7, 8-dihydro-8-oxoguanine by DNA polymerase h. Nat. Genet. 25: dependent upon Rad18 and the other Rad18 indepen- 458–461. dent but Rad5 dependent (Liefshitz et al. 1998; Cejka Haracska, L., I. Unk,R.E.Johnson,E.Johansson,P.M.J.Burgers et al. 2001; Minesinger and Jinks-Robertson 2005). et al., 2001 Roles of yeast DNA polymerases d and z and of Rev1 in the bypass of abasic sites. Genes Dev. 15: 945–954. Since Rad18 is necessary for Rad6 to carry out PCNA Haracska,L.,S.PrakashandL.Prakash,2002 YeastRev1proteinisaG ubiquitination (Hoege et al. 2002), one has to assume template-specific DNA polymerase. J. Biol. Chem. 277: 15546–15551. aracska orres amos ohnson rakash that the Rad18-independent but Rad5-dependent path- H , L., C. A. T -R ,R.E.J ,S.P and L. Prakash, 2004 Opposing effects of ubiquitin conjugation way can function in mutagenesis in the absence of and SUMO modification of PCNA on replicational bypass of DNA PCNA ubiquitination. It is not clear at present how lesions in Saccharomyces cerevisiae. Mol. Cell. Biol. 24: 4267–4274. Polz-dependent TLS through DNA lesions would oc- Hoege, C., B. Pfander, G.-L. Moldovan,G.Pyrowolakis and S. 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