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Originally published as Genetics Published Articles Ahead of Print on August 30, 2008.
Genetics, Vol. 180, 73-82, September 2008, Copyright © 2008
doi:10.1534/genetics.108.091066
Requirement of Rad5 for DNA Polymerase
-Dependent Translesion Synthesis in Saccharomyces cerevisiae
Vincent Pagès*,
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 Intégrité du Génome, UMR7175 Centre National de la Recherche Scientifique/Université Louis Pasteur, Ecole Supérieure de Biotechnologie de Strasbourg, Bld S. Brant, BP 10413, 67412 Illkirch, France and
CNRS, Unité Propre de Recherche 3081, Genome Instability and Carcinogenesis Conventionné par l'Université d'Aix-Marseille 2, 13402 Marseille Cedex 20, France
1 Corresponding author: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1061.
E-mail: l.prakash{at}utmb.edu
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
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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).
|
-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 CAN1s 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.
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Communicating editor: E. ALANI
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