In yeast, Rad6–Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases or 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 Pol. 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 Pol-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.

---

IN the yeast Saccharomyces cerevisiae, the Rad6–Rad18 ubiquitin-conjugating enzyme complex (BAILLY et al. 1994, 1997) promotes replication through DNA lesions by DNA polymerase (Pol) - and -mediated translesion synthesis (TLS) (NELSON et al. 1996b; JOHNSON et al. 1999b; PRAKASH et al. 2005), and by a Rad5–Mms2–Ubc13-dependent pathway in which the gaps that form opposite DNA lesion sites could be filled in by template switching (TORRES-RAMOS et al. 2002; GANGAVARAPU et al. 2006; BLASTYAK et al. 2007). Pol is unique among eukaryotic TLS Pols in its proficient and error-free ability to replicate through UV-induced cyclobutane pyrimidine dimers (CPDs) (JOHNSON et al. 1999b); hence inactivation of Pol in humans and deletion of the yeast RAD30 gene, which encodes Pol, leads to a high incidence of UV mutagenesis (MCDONALD et al. 1997; YU et al. 2001; STARY et al. 2003) and in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum (JOHNSON et al. 1999a; MASUTANI et al. 1999). Although proficient replication through a DNA lesion such as a CPD or an 8-oxoguanine can be mediated by a single TLS Pol, as for example, by Pol (JOHNSON et al. 1999b; HARACSKA et al. 2000), replication through many DNA lesions requires the consecutive action of two different Pols, in which one Pol inserts the nucleotide opposite the lesion site and another Pol carries out the subsequent extension reaction (BRESSON and FUCHS 2002; PRAKASH et al. 2005). Pol, composed of the Rev3 catalytic and Rev7 accessory subunits (NELSON et al. 1996b), plays an important role in TLS by extending from the nucleotide inserted opposite a DNA lesion by another Pol (JOHNSON et al. 2000, 2001, 2003; HARACSKA et al. 2001; PRAKASH et al. 2005; NAIR et al. 2006, 2008).

In the Mms2–Ubc13–Rad5-dependent postreplication repair (PRR) pathway, the Mms2–Ubc13 ubiquitin-conjugating enzyme complex in conjunction with Rad5 carries out the lysine 63-linked polyubiquitination of PCNA at its K164 residue. In DNA damaged yeast cells, PCNA is first monoubiquitinated at the K164 residue by Rad6–Rad18 and subsequently, this lysine residue is polyubiquitinated via the action of the Mms2–Ubc13–Rad5 complex (HOEGE et al. 2002). Rad5, a member of the SWI/SNF family of ATPases (JOHNSON et al. 1992, 1994), exhibits a DNA helicase activity that is highly specialized for promoting replication fork regression (BLASTYAK et al. 2007). Rad5 additionally harbors a C₃HC₄ motif characteristic of ubiquitin ligases (ZACHARIAE et al. 1998; JOAZEIRO et al. 1999; LORICK et al. 1999; FANG et al. 2000). 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 Rad6–Rad18 complex (ULRICH and JENTSCH 2000). Mutational inactivation of the DNA helicase activity or the ubiquitin ligase activity of Rad5 causes the same high degree of defectiveness in the repair of discontinuities that form in the newly synthesized strand in UV-damaged cells as that in the rad5 mutant, indicating that both these activities are important for Rad5 function in PRR (GANGAVARAPU et al. 2006). The direct involvement of Rad5 DNA helicase activity in promoting template switching and thereby enabling the use of the nascent lagging strand as the template for synthesizing DNA complementary to the damaged region provides for an important means by which replication through the lesion site can be accomplished in an error-free manner (BLASTYAK et al. 2007).

As expected from the roles of Rad5 and Pol in promoting error-free synthesis through UV lesions, the frequency of UV-induced forward mutations at the CAN1 locus is greatly enhanced in the rad5 rad30 double mutant compared to that in either single mutant (JOHNSON et al. 1999c). Curiously, however, the loss of Rad5 function adversely affects UV-induced reversion of ochre alleles (LEMONTT 1971; LAWRENCE and CHRISTENSEN 1978; JOHNSON et al. 1992). For example, UV-induced reversion of arg4-17 to Arg+ is reduced 10-fold in the rad5 strain (JOHNSON et al. 1992). Sequence analyses of UV-induced arg4-17 to ARG4+ revertants has indicated the reversion to be predominantly a T C transition of T127 that would constitute the 3'T of a potential TT photoproduct (ZHANG and SIEDE 2002). To delineate whether the function of Rad5 in UV-induced mutations at arg4-17 was mediated in collaboration with Mms2–Ubc13, in a previous study, we examined the incidence of UV-induced reversion of arg4-17 in the mms2, ubc13, and rad5 strains (GANGAVARAPU et al. 2006). However, we found that in this role, Rad5 functioned independently of Mms2–Ubc13. Moreover, the inactivation of Rad5 helicase function or of its ubiquitin ligase function had no adverse affect upon UV mutagenesis. From such observations, we concluded a structural role of Rad5 in UV mutagenesis at sites such as arg4-17. However, from all the previous studies, only a very limited role of Rad5 in UV mutagenesis that is restricted to the reversion of ochre alleles could be inferred.

Recently, we have reported on our analyses of TLS opposite an abasic (AP) site in yeast cells wherein we employed a plasmid system in which bidirectional replication proceeds from a yeast origin of replication (PAGES et al. 2008). We showed that the rate and genetic control of TLS for both the leading and lagging DNA strands is very similar and that Pol and PCNA ubiquitination are indispensable for TLS on both the DNA strands (PAGES et al. 2008). Using this plasmid system, we have now examined the effects of the rad5, mms2, and ubc13 mutations on TLS through the AP site. In addition, we have carried out studies of TLS through a number of other DNA lesions—a cis–syn TT dimer, a (6-4) TT photoproduct, and an AAF adduct—that are also carried on a duplex plasmid. Also in this plasmid, replication initiates from a single-origin site and proceeds through a site-specific DNA lesion (BAYNTON et al. 1998; BRESSON and FUCHS 2002). From all these studies we conclude a requirement of Rad5 for TLS dependent upon Pol. We elaborate upon the possible implications of these observations for Rad5's role in TLS opposite a diverse array of DNA lesions.

Yeast strains:

For the study of AP bypass (Tables 1 and 2), we used strain EMY74.7 (MATa his3-1 leu2-3,-112 trp1 ura3-52) from which the two AP endonuclease genes APN1 and APN2 have been deleted to prevent the repair of the abasic site. Additionally, we deleted the MSH2 gene to prevent the removal of the mismatch loop present opposite the AP site (Figure 1A). We refer to this apn1 apn2 msh2 strain as the wild-type strain since it is wild type with respect to the proteins involved in lesion bypass. The various genomic deletion and other mutations were introduced into this apn1 apn2 msh2 strain (PAGES et al. 2008). For the study of AAF and UV lesion bypass, plasmids containing a single lesion (BAYNTON et al. 1998; BRESSON and FUCHS 2002) were transformed into the yeast strain EMY74.7 or into its rad30, rad5, or rad30 rad5 derivatives (Tables 3–6).

View this table: In this window In a new window   TABLE 1 TLS frequencies opposite an AP site in different yeast strains

 

View this table: In this window In a new window   TABLE 2 Types and frequencies of nucleotides incorporated opposite an AP site in the rev3, rad5, and mms2 strains

 

View larger version (29K): In this window In a new window Download PPT slide   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 Ura+ cells and the frequency of Ura+ cells among Trp+ 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 (+1 and –1 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.

 

View this table: In this window In a new window   TABLE 3 Effects of Pol and Rad5 on error-free and mutagenic TLS opposite a cis–syn TT dimer

 

View this table: In this window In a new window   TABLE 4 Effects of Pol and Rad5 on error-free and mutagenic TLS opposite a (6-4) TT photoproduct

 

View this table: In this window In a new window   TABLE 5 Effects of Pol and Rad5 on TLS through a G-AAF adduct present in a 3'-GTTT sequence

 

View this table: In this window In a new window   TABLE 6 Effects of Pol and Rad5 on TLS through a G-AAF adduct present in a 3'-GCCC sequence

 
To study the requirement of Rad5 in TLS opposite an AP site (Table 1), we used a rad5 strain in which the wild type or its mutant derivatives are carried on a YCplac133-based plasmid, and which contains the ARS1 origin of replication, the centromeric CEN4 region, and the LEU2 gene. The plasmid, pR5-28, expresses the wild-type Rad5 protein (RAD5+). Plasmid pR5-30 carries the mutations D681, E682 AA in RAD5, which inactivate the ATPase and DNA helicase activities of Rad5 (rad5-ATPase mutant), and plasmid pR5-19 carries the mutations C914, C917 AA in the C₃HC₄ ring-finger motif that abolishes the ubiquitin ligase function (rad5-Ub ligase mutant).

Duplex plasmids:

Double-stranded, closed circular plasmids were generated using the gapped-duplex method (BROSCHARD et al. 1999). For the AP-containing plasmid, the damaged strand carries the TRP1 gene that allows for the selection of transformants resulting from the replication of this strand. The AP site is located in a heteroduplex leader sequence that we inserted in the URA3 gene at its 5' end in two different orientations, which thereby presents the AP site on the leading or the lagging DNA strand during replication (Figure 1A). This construct was obtained by the ligation of a 16-mer oligonucleotide 5'-GGAAGCAATXGTACGG-3' (where X denotes a tetrahydrofurane-type AP site) into a gapped-duplex structure. This leader sequence where the damaged oligonucleotide is ligated is in frame with the URA3 gene, whereas the opposite strand contains a +1 frameshift that inactivates the ura3 gene. Hence cells arising from the replication through the AP site by TLS are Ura+, and cells that underwent non-TLS-mediated replication of the damaged strand (such as copy-choice events) are Ura–. All cells resulting from the replication of the damaged strand are Trp+ (Figure 1A). Plasmids containing a single G-AAF adduct, a cis–syn TT dimer, or a (6-4) TT photoproduct (Figure 1B) were constructed as in (BAYNTON et al. 1998; BRESSON and FUCHS 2002). These constructs contain a short sequence heterology opposite the lesion site to be able to monitor TLS (Figure 1C).

Yeast transformation and identification of TLS products:

Plasmids carrying the AP site were introduced into yeast cells by electroporation as previously described (PAGES et al. 2008). Briefly, cells were grown to exponential phase in YPD, washed several times, and concentrated in 1 M sorbitol. Then 20 ng of plasmid DNA were electroporated, and 1 ml of YPD was added. After incubation for 40 min at 30° the cell suspension was washed with water before plating on selective media: either synthetic complete media lacking tryptophan (SC –trp) to select for the cells that had replicated the damaged strand or SC –trp lacking uracil (SC –trp –ura) to select for plasmids that underwent TLS through the AP site. The ratio of Ura+/Trp+ colonies indicated the TLS frequency. To identify the nucleotide inserted opposite the AP site during TLS, Ura+ colonies were analyzed by direct PCR followed by digestion by a restriction enzyme (PAGES et al. 2008). TLS events opposite the cis–syn TT dimer, (6-4) TT photoproduct, and the G-AAF adduct were detected by colony hybridization using strand-specific oligonucleotides (BRESSON and FUCHS 2002). The frequency of mutagenic TLS through the G-AAF adduct was determined by overlaying SC –trp plates with X-Gal-containing agarose (BRESSON and FUCHS 2002). The +1 and –1 frameshift mutations in the GTTT and GCCC context, respectively, restore the lacZ gene reading frame. The molecular nature of the induced mutation was further confirmed by sequencing.

Physical interaction of Rad5 with Rev1 and Pol:

For physical interaction studies, RAD5, REV1, and REV3 genes were inserted into the vector pBJ842 to produce an amino-terminal glutathione-S-transferase (GST) fusion protein. To purify Pol, GST–REV3 protein was coexpressed with Rev7 in yeast strain BJ5464. Proteins were purified on glutathione-sepharose beads by using a protocol described earlier (JOHNSON et al. 2006). To obtain untagged proteins, GST fusion proteins bound to glutathione-sepharose beads were treated overnight at 4° with PreScission protease which cleaved between the GST tag and the protein of interest. The physical interaction of Rad5 with Rev1 and Pol was examined using a protocol similar to that described earlier (ACHARYA et al. 2005). Briefly, GST–Rad5 or GST alone was incubated with Rev1, and GST–Pol was incubated with Rad5 in buffer I (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5mM dithiotheritol, 0.01% NP-40, and 10% glycerol) in a 20 µl reaction at 4° for 30 min, followed by 10 min at 25°. To this mixture, 20 µl glutathione-sepharose beads were added and further incubated for 1 hr with constant rocking at 4°. The beads were spun down and the unbound protein was collected. Further, the beads were washed thoroughly three times with 10 vol of buffer I. Finally, the bound proteins were eluted with 20 µl of SDS loading buffer. Various fractions were resolved on a 12% denaturing polyacrylamide gel, followed by Coomassie blue R-250 staining.

Requirement of Rad5 for TLS opposite an AP site:

The details of the plasmid system that was used for these studies have been described previously (PAGES et al. 2008). Briefly, the plasmid carries a yeast replication origin ARS1 and a site-specific AP site located on the leading or the lagging DNA strand. Since a tetrahydrofuran lesion was used as an AP site analog, there may exist differences in its bypass from that of an AP site. In this system, replication of the AP site-containing DNA strand, regardless of whether AP bypass occurred by TLS or by any other mechanism, such as template switching, results in a Trp+ cell. The AP site is present in a heteroduplex leader sequence in the URA3 gene and cells harboring plasmid resulting from TLS through the AP site are Ura+. The frequency of TLS among the transformants is determined from the ratio of colonies that grow on –trp –ura media vs. those that grow on –trp (Figure 1A).

As shown in Table 1, TLS accounts for 5–6% of AP bypass in wild-type cells, and the frequency of TLS is reduced by 90% in rev3 cells. Interestingly, the frequency of TLS also shows a large reduction in the rad5 strain nearly similar to that in the rev3 strain. To determine whether the Rad5 helicase and the ubiquitin ligase activities contribute to TLS opposite the AP site, we introduced into rad5 cells a plasmid carrying the wild-type RAD5 gene or the rad5 mutant gene inactivated for the helicase or the ubiquitin ligase function. We found that both the helicase-defective and the ubiquitin-ligase-defective rad5 mutant genes restored TLS in rad5 cells to almost the same level as that conferred upon by the wild-type RAD5 gene. Hence both the Rad5 helicase and ubiquitin ligase activities are dispensable for Rad5's role in mediating TLS opposite an AP site. In keeping with the lack of requirement of the ubiquitin ligase activity of Rad5, we find that the mms2 or ubc13 mutations also cause no significant impairment of TLS opposite this lesion site (Table 1).

Sequence analysis of TLS products from wild-type cells has shown that opposite an AP site on both DNA strands an A is incorporated with a frequency of 70%, a C is incorporated with a frequency of 25%, and G and T insertions are much rarer. As shown in Table 2, the predominance of A insertion persists in the rev3 strain and also in the rad5 and mms2 strains. Thus, even though the frequency of TLS is greatly reduced in the rev3 and rad5 strains, the nucleotide insertion pattern remains about the same in these mutant strains as in the wild-type strain.

Rad5 is not required for error-free TLS opposite a cis–syn TT dimer by Pol:

The duplex plasmid system used for determining the role of Rad5 in promoting 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 TLS accounts for 13% of bypass through the TT dimer and almost all of it is error free (Table 3). TLS is reduced by 50% in the rad30 strain and about the same level of reduction occurs in the rad5 strain. Interestingly and importantly, a synergistic decline in TLS frequency occurs in the rad30 rad5 strain such that the level of TLS decreases by >15-fold (Table 3). From these observations we infer a role for Rad5 in promoting TLS through the TT dimer via a pathway that acts independently of Pol.

Requirement of Rad5 for TLS opposite a (6-4) TT photoproduct:

In wild-type yeast cells, TLS accounts for 4% of bypass opposite the (6-4) TT photoproduct, of which 1.6% results from error-free synthesis through the photoproduct and 2.5% results from mutagenic bypass that involves a 3'T C transition (BRESSON and FUCHS 2002) (Table 4, Figure 1C). In the rad30 strain, mutagenic TLS is reduced by >10-fold while the level of error-free TLS is not affected (BRESSON and FUCHS 2002). Similar to that in rad30, the incidence of mutagenic TLS shows a large decrease in the rad5 strain, but unlike that for rad30, the frequency of error-free TLS is also reduced 4-fold in the rad5 strain (Table 4). Thus, whereas the absence of Pol affects only the mutagenic component of TLS, the absence of Rad5 affects the incidence of both the mutagenic and error-free modes of TLS. Moreover, and interestingly, the absence of Rad5 alone has the same adverse effect on TLS as the absence of both Rad5 and Pol, implicating an epistatic relationship of Rad5 with Pol function.

Requirement of Rad5 for TLS opposite a guanine-AAF adduct:

AAF predominantly forms an adduct at the C8 position of guanine (KRIEK et al. 1967). Previously BRESSON and FUCHS (2002) examined the genetic control of TLS through this adduct in two different sequence contexts, a 3'-GTTT sequence, in which the adducted G is followed by 3 T's on the 5' side in the template strand and a 3'-GCCC sequence where the adducted G is followed by 3 C's on the 5' side (Figure 1C). In a wild-type yeast strain, when the adduct is located in the 3'-GTTT sequence, TLS accounts for 6.7% of bypass events, and a great majority of the events are error free (6.5%), resulting from a C insertion opposite the G-AAF adduct, whereas a small proportion of TLS events (0.2%) result from frameshifting of the primer strand which generates a +1 T insertion (Table 5, Figure 1C). In the rad30 strain, the frequencies of both error-free TLS and mutagenic TLS decrease 4-fold. Importantly, we find that in the rad5 strain, the incidence of error-free TLS declines by >30-fold, from 6.5% in the wild-type strain to 0.2% in the rad5 strain, and there is almost a complete absence of mutagenic TLS (Table 5). Furthermore, the magnitude of decline in the incidence of error-free and mutagenic TLS in the rad5 rad30 strain resembles that in the rad5 mutant alone (Table 5), which implicates epistasis of Rad5 over Pol function in mediating TLS opposite the G-AAF adduct.

In the 3'-GCCC context in the wild-type strain, error-free TLS accounts for almost 99% of TLS, the remainder (1%) being mutagenic events resulting from the frameshifting of the template strand that generates a –1 C replication product (Figure 1C). In the rad30 strain, the frequency of error-free TLS drops 10-fold and mutagenic TLS is almost abolished, whereas in the rad5 and the rad5 rad30 strains, no TLS products were recovered (Table 6). Thus, in both sequence contexts, TLS opposite the G-AAF adduct is affected to a much greater degree in the rad5 strain than in the rad30 strain, and Rad5 displays epistasis over Pol action.

Physical interaction of Rad5 with Rev1:

As we elaborate in the DISCUSSION, our genetic observations indicating a requirement of Rad5 for TLS opposite DNA lesions such as an AP site, a (6-4) TT photoproduct, and a G-AAF adduct, support a role for Rad5 in Pol-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 interactions with Pol, or with Rev1, which is a necessary element for Pol function in TLS. We have shown previously that Rev1 forms a physical complex with Pol and have suggested a role for Rev1 in the targeting of Pol to the replication fork stalled at a DNA lesion (ACHARYA et al. 2006).

To check for the physical interaction of Rad5 with Rev1 and with Pol, we bound a mixture of purified GST–Rad5 and Rev1 protein to the glutathione-sepharose beads, rocked it for 1 hr, followed by extensive washings with 150 mM NaCl-containing buffer before eluting it with SDS-containing buffer. In such a system, GST fusion protein will bind to the beads and the interacting protein will be pulled down only if it forms a stable complex. As shown in Figure 2, Rev1 eluted together with Rad5, indicating that Rad5 forms a stable physical complex with Rev1 at physiological salt concentration (Figure 2, lane 4). In the control experiments, Rev1 did not show any interaction with GST protein alone (Figure 2, lane 8). We found no evidence for the interaction of Rad5 with Pol, as indicated from the absence of any Rad5 in the eluate (Figure 2, lane 12). The ability of Rev1 to directly bind Rad5 may provide a means whereby Rev1 targets Pol to the replication fork stalled at the DNA lesion site (see DISCUSSION).

View larger version (29K): In this window In a new window Download PPT slide   FIGURE 2.— Rad5 directly binds to Rev1 but not to Pol. Yeast Rev1 was mixed and incubated with GST–Rad5 (lanes 1–4) or with GST protein alone (lanes 5–8), and Rad5 was mixed and incubated with GST–Pol (Rev3–Rev7) (lanes 9–12). One microgram of each protein was used in this study. After incubation, samples were bound to glutathione-sepharose beads for 1 hr, followed by multiple washings with buffer I containing 150 mM NaCl and elution of the bound proteins with SDS-sample buffer. Aliquots of each sample before addition to the beads (L), the flow through fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on an SDS-12% polyacrylamide gel developed with Coomassie blue.

 

Our analyses of TLS opposite a number of site-specific DNA lesions carried on duplex plasmids have provided strong evidence for the requirement of Rad5 in TLS. We discuss below the implications of these observations for Rad5 involvement in Pol-dependent TLS and consider the possible ways by which Rad5 may act in such a role.

Requirement of Rad5 for TLS mediated by Pol:

AP site:

Here we show that the rad5 mutation confers almost the same high level of defect in TLS opposite an AP site as does the rev3 mutation. Previously, we have shown that Pol is highly inefficient at inserting a nucleotide opposite the AP site but it can proficiently extend from the nucleotide inserted opposite the lesion site by another DNA Pol (HARACSKA et al. 2001). Since an A is the most frequent nucleotide inserted opposite the AP site in our plasmid system, and because the replicative Pols such as Pol and Pol are able to insert an A opposite this lesion, we have previously suggested that following the A insertion by the replicative Pol, Pol performs the extension reaction (HARACSKA et al. 2001). Since Rev1 promotes C insertion opposite the AP site (NELSON et al. 1996a; HARACSKA et al. 2002), which constitutes the second most frequent event (PAGES et al. 2008), that would also be followed by extension by Pol. Overall, because of the possible involvement of multiple Pols at the insertion step, but of only Pol at the extension step, the requirement of Pol for AP bypass would be much more absolute than that of other Pols. Our observation that Rad5 is also required for TLS opposite the AP site would imply that Rad5 is important for Pol's ability to function in TLS opposite this lesion site.

(6-4) TT photoproduct:

TLS opposite this photoproduct either can occur in an error-free way by the insertion of an A opposite the 3'T of the photoproduct by a DNA Pol whose identity remains to be determined or can occur in a mutagenic way by the insertion of a G by Pol opposite this lesion site (JOHNSON et al. 2001; BRESSON and FUCHS 2002). Since Pol is highly inefficient at inserting a nucleotide opposite the 3'T of this lesion, but can carry out efficient extension from the nucleotide inserted opposite this site by another Pol, we have previously suggested that following nucleotide insertion opposite the 3'T site by another Pol, Pol performs the subsequent extension reaction (JOHNSON et al. 2000, 2001). A role for Pol in promoting mutagenic TLS in which a 3'T-to-C change occurs is in accordance with the ability of this Pol to insert a G opposite this lesion site (JOHNSON et al. 2001; BRESSON and FUCHS 2002). Since the level of TLS opposite a (6-4) TT photoproduct in yeast cells is greatly reduced in the rev3 strain (GIBBS et al. 2005), the indispensability of Pol for both the error-free and mutagenic modes of TLS opposite the lesion site would then result from its absolute requirement at the extension step. Our finding that the level of both error-free and mutagenic TLS opposite this lesion site is greatly reduced in the rad5 strain adds further support for the requirement of Rad5 in TLS mediated by Pol.

G-AAF adduct:

Previously we have shown that in yeast cells, TLS opposite this adduct absolutely requires Pol (BAYNTON et al. 1998). Furthermore, Pol makes a very significant contribution to both the error-free and mutagenic modes of TLS opposite this adduct; consequently, a large reduction in both these modes of TLS occurs in the rad30 strain (BRESSON and FUCHS 2002). Here we show that TLS opposite the G-AAF adduct carried in both the sequence contexts is almost abolished in the rad5 strain. We interpret these various observations to suggest that TLS opposite the G-AAF adduct occurs by nucleotide insertion opposite the lesion by Pol or by another Pol followed by extension by Pol. The requirement of Pol as well as of Rad5 for TLS opposite this lesion site reinforces further the need for Rad5 in modulating Pol-dependent TLS.

Rad5 is not required for Pol-mediated TLS opposite a cis–syn TT dimer:

Although our observations with the above-noted lesions have provided clear evidence that Rad5 can be as indispensable for TLS across these lesions as is Pol, they do not exclude the possible requirement of Rad5 for Pol's ability to carry out its role in TLS opposite the (6-4) TT photoproduct or the G-AAF adduct. That is because opposite both these lesions, the effect of the rad5 mutation is much more drastic than that of the rad30 mutation and the action of Pol is subserved under that of Rad5. Hence Rad5 could be indispensable not only for the action of Pol in TLS but also for that of Pol.

Our analyses of TLS opposite a TT dimer, however, clearly point to the lack of involvement of Rad5 in modulating Pol action opposite this lesion site. Opposite a cis–syn TT dimer, Pol would function independently of Pol. Our finding that a synergistic decline in error-free bypass occurs in the absence of both Pol and Rad5 implies that Pol and Rad5 function independently in promoting TLS opposite a TT dimer. Furthermore, since Pol provides the only other TLS pathway in addition to Pol, a role of Rad5 alternate to Pol implies that Rad5 functions in modulating the Pol-dependent error-free bypass through this lesion site where, following the insertion of an A opposite the 3'T of the dimer by a Pol other than by Pol, the subsequent extension reaction would be carried out by Pol.

Structural role of Rad5 in the assembly of Pol at the stalled replication fork:

As determined from the analyses of TLS opposite an AP site, we find that the inactivation of Rad5 helicase activity or of its ubiquitin ligase activity has no significant effect on TLS, and the absence of either Mms2 or Ubc13 also confers no perceptible impairment of TLS. Since neither the DNA helicase nor the ubiquitin ligase activities contribute to Rad5's role in TLS we surmise that Rad5 functions as a structural element in modulating Pol function in TLS.

Previous studies have indicated a role for Rad5 in promoting error-free postreplication repair by template switching where its ubiquitin ligase activity is used for PCNA polyubiquitination and its DNA helicase activity is used for fork regression. Here we suggest a third function for Rad5 wherein as a structural element, it would modulate Pol's function in TLS. How might Rad5 contribute to Pol's role in TLS? Previously we have provided evidence for the formation of a complex between Rev1 and Pol and have shown that this complex formation is important for Pol's function in TLS (ACHARYA et al. 2006). Our observation that Rad5 directly binds to Rev1 but not to Pol raises the possibility that physical association with Rad5 affects the ability of Rev1 to target Pol to the replication fork stalled at a DNA lesion. Two-hybrid analyses and co-immunoprecipitation studies have shown that Rad5 exists in a complex with Rad6–Rad18 in yeast cells and Rad5 binds this complex via Rad18 (ULRICH and JENTSCH 2000). It is quite likely that the Rad6–Rad18–Rad5 complex binds to the template strand at the lesion site and from there it effects PCNA ubiquitination; additionally, this complex could also have a role in promoting the assembly of the TLS Pols at the lesion site. In that case, the binding of Rad5 by Rev1 could be important for the targeting of Pol to the DNA lesion site where replication has stalled.

Other considerations:

The requirement of Rad5 for Pol-dependent TLS that we have uncovered here from the studies done with duplex plasmids carrying a site-specific DNA lesion stands in contrast to the lack of requirement of Rad5 in Pol-mediated TLS in UV-irradiated cells. In fact, in UV-damaged yeast cells, UV-induced forward mutations at the CAN1^s locus arise in rad5 cells and their frequency is further enhanced in the rad5 rad30 strain (JOHNSON et al. 1992, 1999c). These observations have previously been ascribed to a much greater dependence upon the error-prone Pol for lesion bypass when both the error-free pathways—Rad5-dependent template switching and Pol-dependent error-free TLS through the CPDs—have been inactivated. These seemingly disparate observations for the possible requirement of Rad5 in Pol-dependent TLS when the lesion is carried on a duplex plasmid but the cells have not been treated with a DNA damaging agent vs. those carried out in UV-irradiated cells could best be reconciled if we assume that in UV-damaged cells, another protein is able to substitute for Rad5. Presumably, this other protein is nonfunctional in cells not treated with a DNA damaging agent because either it is not expressed or it needs to be activated by a post-translational modification such as phosphorylation, and that occurs only when the cells have sustained significant levels of DNA damage. However, the requirement of Rad5 for UV mutagenesis of certain ochre alleles such as arg4-17 raises the possibility that even though the substitute protein can function in lieu of Rad5 in most of the sequence contexts, the function of Rad5 is still required for modulating Pol-dependent TLS through some sequence regions.

Although in its requirement for Rad5, Pol-dependent TLS through site-specific DNA lesions carried on duplex plasmids in undamaged yeast cells differs from TLS in UV-damaged cells where Rad5 can be dispensable for UV mutagenesis, TLS in both these cases depends upon PCNA ubiquitination (STELTER and ULRICH 2003; HARACSKA et al. 2004; PAGES et al. 2008). The requirement of PCNA ubiquitination for TLS opposite a single DNA lesion in undamaged yeast cells as inferred from plasmid studies has suggested that the stalling of replicative DNA polymerase at the DNA lesion is sufficient to generate a signal for Rad6–Rad18-mediated PCNA ubiquitination and that a certain threshold level of DNA damage is not needed for PCNA ubiquitination to occur (PAGES et al. 2008). In recent biochemical experiments in which processively moving yeast Pol was stalled in the presence of PCNA or monoubiquitinated PCNA by nucleotide omission, exchange with Pol could occur only in the presence of ubiquitinated PCNA and not with unmodified PCNA (ZHUANG et al. 2008). Hence, the available genetic and biochemical evidence supports the view that PCNA ubiquitination provides a key mechanism for polymerase exchange to occur when the replicative polymerase stalls at a DNA lesion or from some other cause.

In contrast to the requirement of Rad6–Rad18-dependent PCNA ubiquitination for TLS as inferred from plasmid studies and from the studies of DNA damage-induced mutagenesis, genetic analyses of Pol-dependent spontaneous mutagenesis in yeast have indicated that it can occur via two separate pathways—one dependent upon Rad18 and the other Rad18 independent but Rad5 dependent (LIEFSHITZ et al. 1998; CEJKA et al. 2001; MINESINGER and JINKS-ROBERTSON 2005). Since Rad18 is necessary for Rad6 to carry out PCNA ubiquitination (HOEGE et al. 2002), one has to assume that the Rad18-independent but Rad5-dependent pathway can function in mutagenesis in the absence of PCNA ubiquitination. It is not clear at present how Pol-dependent TLS through DNA lesions would occur in the absence of PCNA ubiquitination. The lack of requirement for PCNA ubiquitination in the Rad5-dependent pathway of spontaneous mutagenesis might suggest that this pathway handles very different types of DNA lesions than those being studied in plasmid assays or involved in UV mutagenesis.

This work was supported by National Institutes of Health grant CA107650 and by a grant from the Groupement d'Intérêt Public ANR-06-BLAN-0258.

ACHARYA, N., L. HARACKSA, R. E. JOHNSON, I. UNK, S. PRAKASH et al., 2005 Complex formation of yeast Rev1 and Rev7 proteins: a novel role for the polymerase-associated domain. Mol. Cell. Biol. 25: 9734–9740.[Abstract/Free Full Text]

ACHARYA, N., R. E. JOHNSON, S. PRAKASH and L. PRAKASH, 2006 Complex formation with Rev1 enhances the proficiency of yeast DNA polymerase for mismatch extension and for extension opposite from DNA lesions. Mol. Cell. Biol. 26: 9555–9563.[Abstract/Free Full Text]

BAILLY, V., J. LAMB, P. SUNG, S. PRAKASH and L. PRAKASH, 1994 Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes and Dev. 8: 811–820.[Abstract/Free Full Text]

BAILLY, V., S. LAUDER, S. PRAKASH and L. PRAKASH, 1997 Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities. J. Biol. Chem. 272: 23360–23365.[Abstract/Free Full Text]

BAYNTON, K., A. BRESSON-ROY and R. P. P. FUCHS, 1998 Analysis of damage tolerance pathways in Saccharomyces cerevisiae: a requirement for Rev3 DNA polymerase in translesion synthesis. Mol. Cell. Biol. 18: 960–966.[Abstract/Free Full Text]

BLASTYAK, A., L. PINTER, I. UNK, L. PRAKASH, S. PRAKASH et al., 2007 Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression. Mol. Cell 28: 167–175.[Medline]

BRESSON, A., and R. P. P. FUCHS, 2002 Lesion bypass in yeast cells: Pol participates in a multi-DNA polymerase process. EMBO J. 21: 3881–3887.[CrossRef][Medline]

BROSCHARD, T. H., N. KOFFEL-SCHWARTZ and R. P. FUCHS, 1999 Sequence-dependent modulation of frameshift mutagenesis at NarI-derived mutation hot spots. J. Mol. Biol. 288: 191–199.[CrossRef][Medline]

CEJKA, P., V. VONDREJS and Z. STORCHOVA, 2001 Dissection of the functions of the Saccharomcyes cerevsiae RAD6 postreplicative repair group in mutagenesis and UV sensitivity. Genetics 159: 953–963.[Abstract/Free Full Text]

FANG, S., J. P. JENSEN, R. L. LUDWIG, K. H. VOUSDEN and A. M. WEISSMAN, 2000 Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J. Biol. Chem. 275: 8945–8951.[Abstract/Free Full Text]

GANGAVARAPU, V., L. HARACSKA, I. UNK, R. E. JOHNSON, S. PRAKASH et al., 2006 Mms2-Ubc13-dependent and -independent roles of Rad5 ubiquitin ligase in postreplication repair and translesion DNA synthesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 26: 7783–7790.[Abstract/Free Full Text]

GIBBS, P. E. M., J. MCDONALD, R. WOODGATE and C. W. LAWRENCE, 2005 The relative roles in vivo of Saccharomyces cerevisiae Pol, Pol, Rev1 protein and Pol32 in the bypass and mutation inductiion of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer. Genetics 169: 575–582.[Abstract/Free Full Text]

HARACSKA, L., S.-L. YU, R. E. JOHNSON, L. PRAKASH and S. PRAKASH, 2000 Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase . Nat. Genet. 25: 458–461.[CrossRef][Medline]

HARACSKA, L., I. UNK, R. E. JOHNSON, E. JOHANSSON, P. M. J. BURGERS et al., 2001 Roles of yeast DNA polymerases and and of Rev1 in the bypass of abasic sites. Genes Dev. 15: 945–954.[Abstract/Free Full Text]

HARACSKA, L., S. PRAKASH and L. PRAKASH, 2002 Yeast Rev1 protein is a G template-specific DNA polymerase. J. Biol. Chem. 277: 15546–15551.[Abstract/Free Full Text]

HARACSKA, L., C. A. TORRES-RAMOS, R. E. JOHNSON, S. PRAKASH and L. PRAKASH, 2004 Opposing effects of ubiquitin conjugation and SUMO modification of PCNA on replicational bypass of DNA lesions in Saccharomyces cerevisiae. Mol. Cell. Biol. 24: 4267–4274.[Abstract/Free Full Text]

HOEGE, C., B. PFANDER, G.-L. MOLDOVAN, G. PYROWOLAKIS and S. JENTSCH, 2002 RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135–141.[CrossRef][Medline]

JOAZEIRO, C. A., S. S. WING, H. HUANG, J. D. LEVERSON, T. HUNTER et al., 1999 The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiqutin-ligase. Science 286: 309–312.[Abstract/Free Full Text]

JOHNSON, R. E., S. T. HENDERSON, T. D. PETES, S. PRAKASH, M. BANKMANN et al., 1992 Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome. Mol. Cell. Biol. 12: 3807–3818.[Abstract/Free Full Text]

JOHNSON, R. E., S. PRAKASH and L. PRAKASH, 1994 Yeast DNA repair protein RAD5 that promotes instability of simple repetitive sequences is a DNA-dependent ATPase. J. Biol. Chem. 269: 28259–28262.[Abstract/Free Full Text]

JOHNSON, R. E., C. M. KONDRATICK, S. PRAKASH and L. PRAKASH, 1999a hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285: 263–265.[Abstract/Free Full Text]

JOHNSON, R. E., S. PRAKASH and L. PRAKASH, 1999b Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Pol. Science 283: 1001–1004.[Abstract/Free Full Text]

JOHNSON, R. E., S. PRAKASH and L. PRAKASH, 1999c Requirement of DNA polymerase activity of yeast Rad30 protein for its biological function. J. Biol. Chem. 274: 15975–15977.[Abstract/Free Full Text]

JOHNSON, R. E., M. T. WASHINGTON, L. HARACSKA, S. PRAKASH and L. PRAKASH, 2000 Eukaryotic polymerases and act sequentially to bypass DNA lesions. Nature 406: 1015–1019.[CrossRef][Medline]

JOHNSON, R. E., L. HARACSKA, S. PRAKASH and L. PRAKASH, 2001 Role of DNA polymerase in the bypass of a (6-4) TT photoproduct. Mol. Cell. Biol. 21: 3558–3563.[Abstract/Free Full Text]

JOHNSON, R. E., S.-L. YU, S. PRAKASH and L. PRAKASH, 2003 Yeast DNA polymerase zeta () is essential for error-free replication past thymine glycol. Genes Dev. 17: 77–87.[Abstract/Free Full Text]

JOHNSON, R. E., L. PRAKASH and S. PRAKASH, 2006 Yeast and human translesion DNA synthesis polymerases: expression, purification, and biochemical characterization. Methods Enzymol. 408: 390–407.[Medline]

KRIEK, E., J. A. MILLER, W. JUHL and E. C. MILLER, 1967 8-(N-2-fluorenyl-acetamide) guanosine and arylamidation reaction product of guanosine and the carcinogen N-acetoxy-N-2-fluorenylacetamide in neutral solution. Biochemistry 6: 177–182.[CrossRef][Medline]

LAWRENCE, C. W., and R. B. CHRISTENSEN, 1978 Ultraviolet-induced reversion of cyc1 alleles in radiation sensitive strains of yeast. II. rev2 mutant strains. Genetics 90: 213–226.[Abstract/Free Full Text]

LEMONTT, J. F., 1971 Mutants of yeast defective in mutation induced by ultraviolet light. Genetics 68: 21–33.[Free Full Text]

LIEFSHITZ, B., R. STEINLAUF, A. FRIEDL, F. ECKARDT-SCHUPP and M. KUPIEC, 1998 Genetic interactions between mutants of the ‘error-prone’ repair group of Saccharomcyes cerevisiae and their effect on recombination and mutagenesis. Mutat. Res. 407: 135–145.[Medline]

LORICK, K. L., J. P. JENSEN, S. FANG, A. M. ONG, S. HATAKEYAMA et al., 1999 RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Nat. Acad. Sci. USA 96: 11364–11369.[Abstract/Free Full Text]

MASUTANI, C., R. KUSUMOTO, A. YAMADA, N. DOHMAE, M. YOKOI et al., 1999 The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase . Nature 399: 700–704.[CrossRef][Medline]

MCDONALD, J. P., A. S. LEVINE and R. WOODGATE, 1997 The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism. Genetics 147: 1557–1568.[Abstract]

MINESINGER, B. K., and S. JINKS-ROBERTSON, 2005 Roles of RAD6 epistasis group members in spontaneous Pol-dependent translesion synthesis in Saccharomyces cerevisiae. Genetics 169: 1939–1955.[Abstract/Free Full Text]

NAIR, D. T., R. E. JOHNSON, L. PRAKASH, S. PRAKASH and A. K. AGGARWAL, 2006 Hoogsteen base pair formation promotes synthesis opposite the 1,N⁶-ethenodeoxyadenosine lesion by human DNA polymerase . Nat. Struct. Mol. Biol. 13: 619–625.[CrossRef][Medline]

NAIR, D. T., R. E. JOHNSON, L. PRAKASH, S. PRAKASH and A. K. AGGARWAL, 2008 Protein-template directed synthesis across an acrolein-derived DNA adduct by yeast Rev1 DNA polymerase. Structure 16: 239–245.[Medline]

NELSON, J. R., C. W. LAWRENCE and D. C. HINKLE, 1996a Deoxycytidyl transferase activity of yeast REV1 protein. Nature 382: 729–731.[CrossRef][Medline]

NELSON, J. R., C. W. LAWRENCE and D. C. HINKLE, 1996b Thymine-thymine dimer bypass by yeast DNA polymerase . Science 272: 1646–1649.[Abstract]

PAGES, V., R. E. JOHNSON, L. PRAKASH and S. PRAKASH, 2008 Mutational specificity and genetic control of replicative bypass of an abasic site in yeast. Proc. Nat. Acad. Sci. USA 105: 1170–1175.[Abstract/Free Full Text]

PRAKASH, S., R. E. JOHNSON and L. PRAKASH, 2005 Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu. Rev. Biochem. 74: 317–353.[CrossRef][Medline]

STARY, A., P. KANNOUCHE, A. R. LEHMANN and A. SARASIN, 2003 Role of DNA polymerase in the UV mutation spectrum in human cells. J. Biol. Chem. 278: 18767–18775.[Abstract/Free Full Text]

STELTER, P., and H. D. ULRICH, 2003 Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425: 188–191.[CrossRef][Medline]

TORRES-RAMOS, C., S. PRAKASH and L. PRAKASH, 2002 Requirement of RAD5 and MMS2 for post replication repair of UV-damaged DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 22: 2419–2426.[Abstract/Free Full Text]

ULRICH, H. D., and S. JENTSCH, 2000 Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J. 19: 3388–3397.[CrossRef][Medline]

YU, S.-L., R. E. JOHNSON, S. PRAKASH and L. PRAKASH, 2001 Requirement of DNA polymerase for error-free bypass of UV-induced CC and TC photoproducts. Mol. Cell. Biol. 21: 185–188.[Abstract/Free Full Text]

ZACHARIAE, W., A. SHEVCHENKO, P. D. ANDREWS, R. CIOSK, M. GALOVA et al., 1998 Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins. Science 279: 1216–1219.[Abstract/Free Full Text]

ZHANG, H., and W. SIEDE, 2002 UV-induced TC transition of a TT photoproduct site is dependent on Saccharomyces cerevisiae polymerase in vivo. Nucleic Acids Res. 30: 1262–1267.[Abstract/Free Full Text]

ZHUANG, Z., R. E. JOHNSON, L. HARACKSA, L. PRAKASH, S. PRAKASH et al., 2008 Regulation of polymerase exchange between Pol and Pol by monoubiquitinatiion of PCNA and the movement of DNA polymerase holoenzyme. Proc. Natl. Acad. Sci. USA 105: 5361–5366.[Abstract/Free Full Text]

Communicating editor: E. ALANI

International Access Link

Online ISSN: 1943-2361  Print ISSN: 0016-6731
Copyright 2009 by the Genetics Society of America
phone: 412-268-1812   fax: 412-268-1813   email: genetics-gsa{at}andrew.cmu.edu