Nucleic Acids Research, 2002, Vol. 30, No. 3 732-739
© 2002 Oxford University Press
Suppression of genetic defects within the RAD6 pathway by srs2 is specific for error-free post-replication repair but not for damage-induced mutagenesis
Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada
Received September 29, 2001; Revised and Accepted November 30, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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srs2 was isolated during a screen for mutants that could suppress the UV-sensitive phenotype of rad6 and rad18 cells. Genetic analyses led to a proposal that Srs2 acts to prevent the channeling of DNA replication-blocking lesions into homologous recombination. The phenotypes associated with srs2 indicate that the Srs2 protein acts to process lesions through RAD6-mediated post-replication repair (PRR) rather than recombination repair. The RAD6 pathway has been divided into three rather independent subpathways: two error-free (represented by RAD5 and POL30) and one error-prone (represented by REV3). In order to determine on which subpathways Srs2 acts, we performed comprehensive epistasis analyses; the experimental results indicate that the srs2 mutation completely suppresses both error-free PRR branches. Combined with UV-induced mutagenesis assays, we conclude that the Pol
-mediated error-prone pathway is functional in the absence of Srs2; hence, Srs2 is not required for mutagenesis. Furthermore, we demonstrate that the helicase activity of Srs2 is probably required for the phenotypic suppression of error-free PRR defects. Taken together, our observations link error-free PRR to homologous recombination through the helicase activity of Srs2. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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Post-replication repair (PRR) is defined as the cellular process that, in the presence of DNA damage, converts low molecular weight genomic fragments into large molecular weight DNA (1,2). This process is required by the cell to survive an S-phase with unrepaired lethal DNA lesions in the genome; the replication machinery bypasses the damage, and PRR processes fill in the resulting gaps. The gap filling event, or damage tolerance, does not remove the lesion, but allows replication to occur in its presence. The mechanism by which PRR functions to resume replication after encountering a block and to fill in the single stranded regions is largely unknown.
In Saccharomyces cerevisiae, members of the RAD6 pathway are responsible for PRR. Rad6 and associated PRR proteins Rad18 and Rev3 were initially thought to mediate PRR via an error-prone mechanism whereby Rad6 and Rad18 promote the activity of Pol (consisting of two subunits Rev3 and Rev7). Pol is a non-essential mutagenic polymerase that is able to replicate over damaged regions of DNA with low fidelity (3), a process referred to as translesion DNA synthesis (TLS). However, many lines of evidence later suggested that the error-prone mechanism of PRR is not the preferred form of tolerance in eukaryotes (for a recent review see 4). The existence of an error-free PRR pathway was suspected but has not been explored until recently. It is now clear that there are at least two independent error-free PRR pathways mediated by RAD5 and POL30, both under the control of RAD6/RAD18 (5), and genes encoding a novel ubiquitin conjugating enzyme complex, Ubc13–Mms2 (6,7), promote one (8) or both (5) of the pathways. RAD5 encodes an ATPase with zinc-finger/RING finger domains (9,10) and physically interacts with Rad18 as well as Ubc13 (8). The activity of this complex is unknown. The pol30-46 allele of the yeast PCNA homolog is sensitive to UV, synergistic with rev3 and defective in PRR (11). The suspected involvement of Pol30 in PRR is not surprising, as PRR and replication are closely related processes. Another member of the PRR epistasis group is RAD30. RAD30 encodes a non-essential DNA polymerase Pol
(12) and is believed to function in error-free translesional bypass (13). RAD30 belongs to the RAD6 epistasis group (13) but its genetic relationship with other members within this pathway remains unclear (5).
Further elucidation of the molecular mechanisms of PRR requires investigation of the genetic relationship between the RAD6 group and other cellular DNA repair/tolerance pathways. Attempts to isolate suppressors of the DNA damage-sensitivity of both rad6 and rad18 null mutants (14–16) led to the identification of SRS2 (RADH), encoding a non-essential DNA helicase with homology to Escherichia coli Rep and UvrD helicases (15). srs2 single mutants display a moderate sensitivity to a wide variety of DNA-damaging agents; the UV sensitivity of the srs2 haploid mutant appears to be restricted to irradiation in the G1-phase of the cell cycle (15).
Recombination repair in S.cerevisiae is crucial for the repair of double stranded breaks, and consists of two subpathways: homologous recombination repair and non-homologous end joining (17). The ability of isolated srs2 alleles to suppress the radiation (UV and 
-ray) sensitivity of rad6 and rad18 cells apparently requires a functional RAD52-dependent homologous recombination repair pathway (16).
The models proposed to explain the relationship between SRS2, the RAD52 pathway and the RAD6 pathway, involve Srs2 as a ‘switch’ (4). The hypothesis that Srs2 functions as a switch between recombination repair and RAD6-dependent PRR is supported by at least two pieces of evidence: (i) suppression of the extreme rad6 and rad18 sensitivity to various DNA-damaging agents is dependent on the RAD52 recombination pathway and (ii) srs2 mutants are hyper-recombinant (18,19); Srs2 is thus considered essential to commit the cell to RAD6-dependent PRR.
In view of the complexity of the RAD6 pathway, it is important to address three major issues: (i) genetic interactions and molecular events within error-free PRR, (ii) connection between error-free PRR and homologous recombination and (iii) mechanism(s) of srs2 suppression of PRR activity (4). We hypothesize that the precise placement of SRS2 function with respect to the three independent branches of RAD6 pathway would provide clues to all the above issues. We report here that the srs2 mutation is able to completely suppress DNA damage sensitivities of the mutants representing both error-free branches within PRR. It appears that the TLS activities within the RAD6 pathway are less dependent on Srs2, although genetic interactions between SRS2 and REV3 have been observed. Finally, we demonstrate that the helicase activity of Srs2 is absolutely required for its function within PRR. Taken together, our results suggest that the Srs2 helicase acts at the level of error-free PRR probably via its control of homologous recombination.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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Strains and cell culture
The S.cerevisiae strains used in this study are listed in Table 1. Strains PY39-0 and PY39-46 (11) were obtained from Dr P. Burgers (Washington University, St Louis). Cogenic strains HK578-10D (wild-type), HK578-6B (rad5
::URA3), HK590-6D (srs2::HIS3) and HK7302 containing the srs2-101 allele (19) were kindly provided by Dr H. Klein (New York University, New York). Construction of the isogenic rev3 (20), mms2 (6), rad5 and rad30 (5) mutants were as described previously. To delete the SRS2 gene from various strains, plasmid pJH684 was obtained from Dr J. Haber (Brandeis University, MA) and the srs2::LEU2 disruption cassette was released by PstI digestion prior to yeast transformation.
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Yeast cells were cultured at 30°C in either rich YPD medium or in a synthetic minimal SD medium supplemented with amino acids and bases (21). A standard yeast transformation protocol was followed with modifications (22).
Determination of UV and methyl methanesulfonate sensitivities
Methyl methanesulfonate (MMS)-induced liquid killing was performed as described previously (20). Briefly, cells were grown overnight in YPD media; 0.5 ml of overnight culture was subcultured into 5 ml of fresh YPD, and allowed to grow until a mid-logarithmic phase was achieved. A 1 ml sample of culture was removed, diluted and plated on YPD with a plating dilution of 10–5; these cells represent the untreated control cells. At this time, MMS was added to the remaining culture to final concentrations as indicated in each experiment. At given time intervals, 1 ml of cells was removed, washed twice with sterile water, diluted to an appropriate concentration and plated in duplicate on YPD plates. Plates were incubated for 3 days at 30°C.
For UV treatment, cells were plated at different dilutions and then exposed to 254 nm UV light, either in a UV crosslinker (Fisher Sci. model FB-UVXL-1000 at 
2400 µW/cm2) or with a UV lamp (UVP model UVGL-25 at 40 µW/cm2) at specified doses. Cells were plated in duplicate on YPD to score cell survival, and the plates were incubated at 30°C for 3 days. Irradiation and the subsequent incubation of the cells were performed in the dark to prevent photoreactivation.
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Gradient plate assay |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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Thirty milliliters of molten YPD agar was mixed with the desired concentration of MMS, and was poured into the bottom of a tilted square Petri dish. After solidification, the Petri dish was returned to level and 30 ml of molten top agar added. An aliquot (0.1 ml) of overnight culture was mixed with 0.9 ml of molten 1% agar and imprinted down the length of the gradient. Plates were incubated at 30°C and monitored for growth.
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Spontaneous mutagenesis assay |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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DBY747 bears a trp1-289 amber mutation that can revert to Trp+ by several different mutational events. Spontaneous Trp+ reversion rates of DBY747 derivatives were measured by a modified Luria and Delbruck fluctuation test as described (23). An overnight yeast culture was used to inoculate five tubes, each containing 10 ml of fresh YPD, to a final titer of 20 cells/ml. Incubation was continued until a titer of 2 x 108 cells/ml was reached. Cells were collected, washed, resuspended and plated. Each set of experiments contained five independent cultures of each strain; each culture was plated onto YPD in duplicate to score total survivors, and onto SD medium lacking tryptophan (SD-Trp) plates to score Trp+ revertants. Spontaneous mutation rates (number of revertants per cell per generation) were calculated as described previously (24). To calculate the frequency of spontaneous mutagenesis the following formula was used:
frequency (F) = total cell number of TRP+ cells/total number of viable cells.
To calculate the rate of spontaneous mutagenesis, the following formula was used:
rate = 0.4343 x F/log(total cell number) – log(initial cell number).
The formula was derived to determine the mutation rate for a replicating system, where 0.4343 represents the logarithm of e.
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UV-induced mutagenesis assay |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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An adaptation of the method of Watkins et al. (25) was used to assess the UV-induced Trp+ reversion. DBY747 and its isogenic derivatives carrying trp1-289 were grown overnight at 30°C in 3 ml of YPD. An aliquot (100 µl) of the overnight culture was subcultured into 5 ml of fresh YPD media. When the culture reached mid-logarithmic growth, serial dilutions were made and cells were plated onto YPD to obtain colony-forming units. Undiluted cultures were plated onto SD-Trp plates to determine Trp+ reversion. Both the YPD and SD-Trp plates were exposed to UV in the dark using the UV lamp. The plates were then incubated for 3 days at 30°C in a dark chamber to prevent photoreactivation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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srs2 is epistatic to mms2
The isolation and genetic characterization of MMS2 (6) provides a key handle to further investigate the error-free PRR pathway. Deletion of MMS2 results in a significant increase in spontaneous mutagenesis, and this increase is dependent on the functional REV3 gene (6). Furthermore, although mms2 and rev3 single mutants display only moderately increased sensitivity to various DNA-damaging agents, the mms2 rev3 double mutant is extremely sensitive to these agents and the effect of double mutation is clearly synergistic (26). These findings established a two-parallel pathway model and placed MMS2 in the error-free PRR branch. Given the strong link between SRS2 and homologous recombination and the assumption that error-free PRR utilizes a recombination mechanism to bypass replication blocks (4), we proposed that srs2 might act to suppress the error-free branch of PRR. Epistasis analysis was performed between srs2 and mms2 in order to see if the suppression extended to the subpathways. As seen in Figure 1, srs2 cells display a similar (Fig. 1A) or a greater (Fig. 1B) sensitivity to UV and MMS, respectively, compared with the mms2 single mutant. In both cases, the srs2 mms2 double mutant is as sensitive as the srs2 single mutant, indicating that srs2 is epistatic to mms2. The fact that the srs2 mms2 double mutant is no more sensitive than either of the corresponding single mutants clearly places these two genes in the same genetic pathway. However, as mms2 is no more sensitive to killing by UV and MMS than the srs2 mutant, one cannot demonstrate a rescuing/suppressing effect of mms2 phenotype as observed with rad6 and rad18 mutants (14–16). One is also unable to address whether Srs2 acts upstream of Rad6 and is required for all PRR, or if it is involved specifically within error-free PRR. We reasoned that if the latter is true, the srs2 mutation should have no effect on the increased mutagenesis observed in mms2 cells. As seen in Figure 2, while the mms2 single mutation causes an increase in the spontaneous reversion frequency of a trp1-289 amber mutation up to 30-fold over wild-type levels, the srs2 mutation completely suppresses the mms2 mutator phenotype to the srs2 single mutant level. This phenomenon is reminiscent of the rev3 mutation. Thus, we conclude that srs2 is indeed a suppressor of mms2, that the mutator phenotype observed in mms2 cells is dependent on both REV3 and SRS2 and that the srs2 suppressor is probably not specific for mms2.
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srs2 is epistatic to both branches of error-free PRR
The error-free PRR activity has been divided into two independent subpathways represented by RAD5 and POL30 (5). MMS2 and UBC13 may be involved in only RAD5 (8) or both (5) subpathways. Hence, it is important to determine whether srs2 suppresses one or both subpathways of error-free PRR. This effect can be directly assessed as we have shown previously that both rad5 and pol30-46 mutants are much more sensitive to UV and MMS than mms2 (5). As shown in Figure 3A and B, rad5 and pol30-46 single mutants are indeed much more sensitive to MMS than the srs2 single mutant. In both cases, the corresponding srs2 double mutants are as sensitive to MMS as the srs2 single mutant. A similar result has been obtained with UV treatment (data not shown). These results support a hypothesis that SRS2 is required for both subpathways of error-free PRR. Furthermore, the extreme MMS sensitivity of the pol30-46 rad5 double mutant is also completely suppressed by SRS2 deletion, with the triple mutant exhibiting MMS sensitivity similar to the srs2 single mutant (Fig. 3C). Together, these results strengthen the hypothesis that the entire error-free PRR (regardless of the genetic relationship of RAD5, POL30 and MMS2/UBC13) is controlled by Srs2.
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RAD30 is an additional member within the RAD6 pathway and, because of its phenotypes with UV-induced mutagenesis, was assigned to error-free PRR (13). Although we have confirmed that rad18 is indeed epistatic to rad30 with respect to UV-induced killing, the rad30 mutant does not display an increased spontaneous mutagenesis, nor is it synergistic with rev3 (27; B.Chow and W.Xiao, unpublished observations). Epistasis analysis with srs2 (Fig. 4) indicates that the srs2 rad30 double mutant is more sensitive to UV-induced killing than either of the corresponding single mutants, but is less sensitive than the expected additive effect (Fig. 4A). Similarly, the rad30 single mutant displays too little sensitivity to killing by MMS (Fig. 4B) to allow any meaningful conclusions.
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SRS2 and Pol
mutagenesisKnowing that srs2 is epistatic to all error-free PRR mutations, and that srs2 suppresses the elevated spontaneous mutagenesis of mms2 mutants, we wished to determine the genetic interactions between SRS2 and the REV1, REV3, REV7 mutagenesis pathway. We chose the rev3 mutation as REV3 encodes the catalytic subunit of DNA Pol
(3) and is required for a broad range of mutagenesis (28,29).
One concern over the epistasis analysis of srs2 and rev3 is that rev3 displays very moderate sensitivity to DNA-damaging agents. Nevertheless, its sensitivity to MMS at a high dose is significant and the srs2 rev3 double mutant appears to be weakly additive (Fig. 5). This result does not allow us to draw definitive conclusions, and may reflect a complicated genetic interaction between SRS2 and REV mutagenesis. To further address the issue, we performed two sets of experiments. First, we took advantage of the strong synergism between rev3 and mms2 (6,26) and wanted to know whether srs2 is able to suppress the extreme sensitivity of the mms2 rev3 double mutant. As shown in Figure 6 of a gradient plate assay, compared with the isogenic wild-type cells (lane 1), mms2 (lane 2) is slightly more sensitive to MMS, rev3 (lane 3) does not display a noticeable sensitivity, whereas the mms2 rev3 (lane 5) double mutant is extremely sensitive. Deletion of SRS2 in the double mutant rescued it from killing by MMS to a level indistinguishable from that of srs2 single mutant (compare lanes 4 and 6). This result appears to favor a notion that srs2 suppresses all three subpathways within the RAD6 group. However, it does not rule out the possibility that simple suppression of the mms2 single mutation in a mms2 rev3 strain is sufficient to result in the observed phenotype, as the rev3 single mutation has little effect on MMS-induced killing. Our second experiment was to take advantage of the strong anti-mutagenic effect of the rev3 mutation, as the rev3 mutant was initially isolated by its lack of UV-induced mutagenesis (30,31). Hence, we examined the effects of rev3 and srs2 mutations on UV-induced mutagenesis by a Trp+ reversion assay. As shown in Table 2, the mutation frequency increased in both wild-type and srs2 cells to a similar extent and in a dose-dependent manner after low-dose UV treatment. In contrast, the rev3 mutation abolished basal and UV-induced mutagenesis in both wild-type and srs2 cells. These results indicate that in the absence of Srs2, Pol is still capable of TLS and that rev3 is epistatic to srs2 with respect to UV-induced mutagenesis.
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The helicase activity of Srs2 is probably required for PRR
The biochemical activity of Srs2 within PRR has not been assigned. In vitro experiments have shown that Srs2 possesses 3' to 5' helicase activity (32), which was shown to be crucial for recombination (19). To assess whether this helicase activity is also essential for the PRR function of Srs2, epistasis analysis was performed using a putative srs2 helicase mutation (srs2-101) in combination with mms2 and rad5 mutations. srs2-101 encodes a Srs2 protein with a single P37L amino acid substitution (19). This mutation is in the conserved domain I, the ATP-binding domain present in all DNA helicases (33). The P37 residue is within the helicase consensus sequence of (A/P)GXGK(S/T) (34) and is expected to be defective in the helicase activity. We found that with respect to UV- and MMS-induced killing, srs2-101 is as sensitive as the srs2
null mutant, is epistatic to both mms2 and rad5 and is able to suppress the extreme sensitivity of rad5 to the same extent as the srs2 null mutant (data not shown). These results are consistent with the srs2-101 suppression of rad18 mutants (19). Therefore, the helicase activity of Srs2, and probably its role(s) in homologous recombination, is essential for its in vivo function in PRR. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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srs2 suppresses mutations in both error-free PRR subpathways
It has been well established that SRS2 is required for the RAD6/RAD18 pathway (14–16) and that SRS2 acts to channel lesions to the PRR pathway (16). We have reported previously that there are at least three rather independent branches within the RAD6 pathway; they probably perform either complementary or competitive functions within the stalled replication fork due to DNA damage (5). However, while the suppression of rad6/rad18 null mutants by srs2 is significant and obvious, the double mutants were often more sensitive to UV than the srs2 single mutant (14–16,19). This led us to hypothesize that srs2 may suppress one or two branches within, instead of the entire RAD6 pathway. To test this hypothesis, we studied the genetic interactions between SRS2 and the other members of PRR. We found that srs2 suppresses mms2
, rad5 and pol30-46 to the extent that all the double mutants were as sensitive to UV and MMS as the srs2 single mutant in their respective strain backgrounds. In other words, in the absence of functional SRS2, both branches of error-free PRR are dispensable without noticeable phenotypic alterations. Our observation is consistent with a recent report that srs2 suppresses rad5 and mms2/ubc13 (35); however, it does not agree with the additive effect between srs2 and pol30-46 in that report. We noticed significantly different levels of sensitivity of pol30-46 strains between the two reports, compared with the respective rad5 and srs2 mutants, which may underlie the two different results. While our pol30-46 mutant was much more sensitive to UV and MMS than srs2 (this study) and was comparable with that of rad5 (5), the pol30-46 mutant created by Ulrich (35) appears to be as sensitive as the srs2 single mutant. To further confirm the epistatic relationship between srs2 and pol30-46, we created the pol30-46 rad5 srs2 triple mutant and found that it was still as sensitive as its isogenic srs2 single mutant. We argue that if srs2 was only involved in the RAD5 subpathway, the srs2 mutation would not completely suppress pol30-46 rad5 cells to the level of a srs2 single mutant. Hence, our results support the notion that Srs2 is involved in both error-free subpathways of PRR.
Srs2 and the mutagenic response
In both this and another study (35), the less-than-additive or even epistatic effects, with respect to killing by DNA-damaging agents, observed between srs2 and rev3 make it difficult for us to draw any meaningful conclusions as to whether srs2 suppresses the rev3 mutation. We reasoned that this ambiguous epistatic relationship might reflect the complexity of the RAD6 pathway and the molecular events yet to be elucidated. To further investigate this issue, we examined whether srs2 can rescue the extreme sensitivity of the mms2 rev3 double mutant. Although the complete phenotypic suppression of the double mutation by srs2 would support that srs2 is epistatic to both mms2 and rev3, a conclusion that is in contrast to the previous report (35), we wish to entertain an alternative possibility that this suppression is specific to mms2 and that the resulting rev3 srs2 double mutant may be indistinguishable from the srs2 single mutant. The UV-induced mutagenesis assay clearly indicates that while REV3 is absolutely required for damage-induced mutagenesis, SRS2 is not required for this process, as the srs2 mutant displays a wild-type level of mutagenesis. rev3 is actually epistatic to srs2 with respect to induced mutagenesis, which again indicates that Srs2 acts somewhere upstream of Pol but is not required for TLS. This result is consistent with a previous report (16) that although srs2 is epistatic to rad6 over killing by DNA-damaging agents, the deletion of RAD6 actually suppresses UV-induced mutagenesis observed in both wild-type and srs2 mutants. To reconcile the seemingly contradictory results, we attempt to entertain two alternative models. The first model proposes that Srs2 prevents the recombination pathway from access to stalled replication forks and indirectly promotes Rad6-dependent PRR. When error-free PRR is inactive, Srs2 will force stalled forks to be resolved by TLS. This model predicts that srs2 and rev3 are additive or even synergistic; however, the observed less-than-additive effect may reflect the interference of Pol access by Srs2 or other unknown factors. The second model proposes that Srs2 promotes all three branches of the RAD6 pathway but prefers the error-free process. Under conditions where error-free PRR is inactive, Srs2 promotes Pol-mediated TLS. This model predicts that srs2 is epistatic to rev3, which would be consistent with some observations (35; and this study).
Contrary to the results obtained from induced mutagenesis, there is little doubt that SRS2 is involved in spontaneous mutagenesis. srs2 limits the elevated spontaneous mutation rates observed in rad18 and rad5 (36) as well as mms2 (this study) mutants. These observations are reminiscent of the suppression of spontaneous mutagenesis by rev3 (6,26) and would support a notion that both Srs2 and Pol are required for spontaneous mutagenesis. The opposite effects of srs2 on spontaneous and induced mutagenesis testify to the complexity of the RAD6 pathway. However, it would be consistent with observations that although rad6 and rad18 are mutators (37,38), they are defective in UV-induced mutagenesis (39,40). While the UV-induced mutagenesis has been traditionally taken as a criterion to determine the involvement of TLS (40), the molecular events affecting the processing of UV-induced lesions may be different from certain spontaneous mutagenesis events that are claimed to be PRR-independent (41). An even more interesting possibility is that Srs2 may switch its roles in spontaneous versus induced mutagenesis. In this regard, POL32, a new member of RAD6 group encoding a non-essential subunit of Pol
(42) that interacts with both Pol30 (42) and Srs2 (43), may provide a missing link. pol32 is synergistic with mms2 and epistatic to rev3, suggesting that it belongs to the error-prone branch; however, pol32 suppresses UV-induced mutagenesis but variably affects spontaneous mutagenesis (43; M.Hanna and W.Xiao, unpublished results). It is conceivable that Pol32 may serve as a coupling factor between replication and TLS in a manner as Srs2 does between stalled replication folks and error-free PRR. It would be more interesting if Pol32 coordinates between Srs2 and Pol (via Pol30/PCNA) at the damage site. Obviously the molecular events leading to the roles played by Srs2 and Pol32 need further investigation.
Srs2 helicase links PRR and recombination
Escherichia coli possesses a tolerance pathway, which predominately uses recombination events to bypass irreparable lesions during replication. Although several observations suggest that recombination participates in PRR, an association between recombination and PRR in eukaryotes has not been clearly established. The phenotypes of srs2 probably provide most of the indirect evidence that links error-free PRR to recombination.
SRS2 appears to be multifunctional: it is involved in PRR (14–16), homologous recombination (16,18,19,44,45) and cell-cycle regulation (46). Furthermore, the srs2 mutation is synthetic lethal or causes a poor growth phenotype with mutations in a number of genes, including RAD50 (19,45), RAD54 (45), SGS1 (47,48), MRE11, XRS2, TOP3 and RAD27 (49). To determine whether the helicase activity of Srs2 is essential for its role(s) in PRR, we utilized a putative helicase-specific mutant known to be hyper-recombinant. This mutation is able to fully rescue mms2 and rad5 null mutants to a level indistinguishable from the srs2 null mutant. Hence, the loss of Srs2 helicase activity appears to result in both stimulation of gene conversion (19) and suppression of error-free PRR. In this regard, it is reasonable to propose that in the presence of damage-induced replication-blocking lesions, the Srs2 helicase activity acts to produce a DNA structure or intermediate recognizable by member(s) in the RAD6 pathway but inaccessible to recombination proteins. This process would be preferred by cells before reaching the 2N stage of the mitotic cell cycle. When the Srs2 helicase activity is inactive, some of the lesions will be processed by homologous recombination, which results in increased gene conversion. However, in the PRR pathway mutants such as rad5, rad6, rad18, pol30-46 and mms2/ubc13 rev3, neither PRR nor homologous recombination is able to process the replication blocks, leading to the extreme cellular sensitivity to DNA-damaging agents. Under this condition, inactivation of srs2 leads to lesion flow to the RAD52 recombination pathway and suppression of the PRR mutant phenotype. Hence, while Srs2 appears to act as a master switch between error-free PRR and recombination, and indirectly influence TLS, it becomes more interesting to find out how the Srs2 helicase activity is regulated during the normal cell cycle and in response to DNA damage.
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ACKNOWLEDGEMENTS |
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The authors wish to thank Drs P. Burgers, J. Haber and H. Klein for yeast strains and plasmids, B. Chow for technical assistance, M. Hanna for preparing the manuscript and other laboratory members for helpful discussion. This work was supported by the Canadian Institutes of Health Research operating grant MOP-38104 to W.X.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 306 966 4308; Fax: +1 306 966 4311; Email: wei.xiao{at}usask.caPresent address:Stacey Broomfield, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2S2, Canada
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSGradient plate assaySpontaneous mutagenesis assayUV-induced mutagenesis assayRESULTSDISCUSSIONREFERENCES |
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1 di Caprio,L. and Cox,B.S. (1981) DNA synthesis in UV-irradiated yeast. Mutat. Res., 82, 69–85.[Web of Science][Medline]
2 Prakash,L. (1981) Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations. Mol. Gen. Genet., 184, 471–478.[Web of Science][Medline]
3 Nelson,J.R., Lawrence,C.W. and Hinkle,D.C. (1996) Thymine-thymine dimer bypass by yeast DNA polymerase . Science, 272, 1646–1649.[Abstract]
4 Broomfield,S., Hryciw,T. and Xiao,W. (2001) DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat. Res., 486, 167–184.[Web of Science][Medline]
5 Xiao,W., Chow,B.L., Broomfield,S. and Hanna,M. (2000) The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways. Genetics, 155, 1633–1641.
6 Broomfield,S., Chow,B.L. and Xiao,W. (1998) MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc. Natl Acad. Sci. USA, 95, 5678–5683.
7 Hofmann,R.M. and Pickart,C.M. (1999) Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell, 96, 645–653.[Web of Science][Medline]
8 Ulrich,H.D. and Jentsch,S. (2000) Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J., 19, 3388–3397.[Web of Science][Medline]
9 Johnson,R.E., Henderson,S.T., Petes,T.D., Prakash,S., Bankmann,M. and Prakash,L. (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.
10 Johnson,R.E., Prakash,S. and Prakash,L. (1994) Yeast DNA repair protein RAD5 that promotes instability of simple repetitive sequences is a DNA-dependent ATPase. J. Biol. Chem., 269, 28259–28262.
11 Torres-Ramos,C.A., Yoder,B.L., Burgers,P.M., Prakash,S. and Prakash,L. (1996) Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair. Proc. Natl Acad. Sci. USA, 93, 9676–9681.
12 Johnson,R.E., Prakash,S. and Prakash,L. (1999) Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Pol. Science, 283, 1001–1004.
13 McDonald,J.P., Levine,A.S. and Woodgate,R. (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]
14 Lawrence,C.W. and Christensen,R.B. (1979) Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants. J. Bacteriol., 139, 866–887.
15 Aboussekhra,A., Chanet,R., Zgaga,Z., Cassier-Chauvat,C., Heude,M. and Fabre,F. (1989) RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. Nucleic Acids Res., 17, 7211–7219.
16 Schiestl,R.H., Prakash,S. and Prakash,L. (1990) The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway. Genetics, 124, 817–831.[Abstract]
17 Paques,F. and Haber,J.E. (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev., 63, 349–404.
18 Aguilera,A. and Klein,H.L. (1988) Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics, 119, 779–790.
19 Rong,L., Palladino,F., Aguilera,A. and Klein,H.L. (1991) The hyper-gene conversion hpr5-1 mutation of Saccharomyces cerevisiae is an allele of the SRS2/RADH gene. Genetics, 127, 75–85.[Abstract]
20 Xiao,W., Chow,B.L. and Rathgeber,L. (1996) The repair of DNA methylation damage in Saccharomyces cerevisiae. Curr. Genet., 30, 461–468.[Web of Science][Medline]
21 Sherman,F., Fink,G.R., Hicks,J. (1983) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
22 Hill,J., Donald,K.A., Griffiths,D.E. and Donald,G. (1991) DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res., 19, 5791.
23 Von Borstel,R.C. (1978) Measuring spontaneous mutation rates in yeast. Methods Cell Biol., 20, 1–24.[Medline]
24 Williamson,M.S., Game,J.C. and Fogel,S. (1985) Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of pms1-1 and pms1-2. Genetics, 110, 609–646.
25 Watkins,J.F., Sung,P., Prakash,S. and Prakash,L. (1993) The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation. Genes Dev., 7, 250–261.
26 Xiao,W., Chow,B.L., Fontanie,T., Ma,L., Bacchetti,S., Hryciw,T. and Broomfield,S. (1999) Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae. Mutat. Res., 435, 1–11.[Web of Science][Medline]
27 Roush,A.A., Suarez,M., Friedberg,E.C., Radman,M. and Siede,W. (1998) Deletion of the Saccharomyces cerevisiae gene RAD30 encoding an Escherichia coli DinB homolog confers UV radiation sensitivity and altered mutability. Mol. Gen. Genet., 257, 686–692.[Web of Science][Medline]
28 Lawrence,C.W. and Christensen,R.B. (1979) Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast. III. rev3 mutant strains. Genetics, 92, 397–408.
29 Lawrence,C.W., O’Brien,T. and Bond,J. (1984) UV-induced reversion of his4 frameshift mutations in rad6, rev1 and rev3 mutants of yeast. Mol. Gen. Genet., 195, 487–490.[Web of Science][Medline]
30 Lemontt,J.F. (1977) Pathways of ultraviolet mutability in Saccharomyces cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance. Mutat. Res., 43, 339–355.[Web of Science][Medline]
31 Lemontt,J.F. (1977) Pathways of ultraviolet mutability in Saccharomyces cerevisiae. III. Genetic analysis and properties of mutants resistant to ultraviolet-induced forward mutation. Mutat. Res., 43, 179–204.[Web of Science][Medline]
32 Rong,L. and Klein,H.L. (1993) Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae. J. Biol. Chem., 268, 1252–1259.
33 Hodgman,T.C. (1988) A new superfamily of replicative proteins. Nature, 333, 22–23.[Medline]
34 Burgess,S., Couto,J.R. and Guthrie,C. (1990) A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing. Cell, 60, 705–717.[Web of Science][Medline]
35 Ulrich,H.D. (2001) The srs2 suppressor of UV sensitivity acts specifically on the RAD5- and MMS2-dependent branch of the RAD6 pathway. Nucleic Acids Res., 29, 3487–3494.
36 Liefshitz,B., Steinlauf,R., Friedl,A., Eckardt-Schupp,F. and Kupiec,M. (1998) Genetic interactions between mutants of the ‘error-prone’ repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis. Mutat. Res., 407, 135–145.[Web of Science][Medline]
37 Kang,X.L., Yadao,F., Gietz,R.D. and Kunz,B.A. (1992) Elimination of the yeast RAD6 ubiquitin conjugase enhances base-pair transitions and G.C-T.A transversions as well as transposition of the Ty element: implications for the control of spontaneous mutation. Genetics, 130, 285–294.[Abstract]
38 Kunz,B.A., Kang,X.L. and Kohalmi,L. (1991) The yeast rad18 mutator specifically increases G.C-T.A transversions without reducing correction of G-A or C-T mismatches to G.C pairs. Mol. Cell. Biol., 11, 218–225.
39 Lawrence,C. (1994) The RAD6 DNA repair pathway in Saccharomyces cerevisiae: what does it do and how does it do it? Bioessays, 16, 253–258.[Web of Science][Medline]
40 Prakash,S., Sung,P. and Prakash,L. (1993) DNA repair genes and proteins of Saccharomyces cerevisiae. Annu. Rev. Genet., 27, 33–70.[Web of Science][Medline]
41 Quah,S.K., von Borstel,R.C. and Hastings,P.J. (1980) The origin of spontaneous mutation in Saccharomyces cerevisiae. Genetics, 96, 819–839.
42 Gerik,K.J., Li,X., Pautz,A. and Burgers,P.M. (1998) Characterization of the two small subunits of Saccharomyces cerevisiae DNA polymerase . J. Biol. Chem., 273, 19747–19755.
43 Huang,M.E., de Calignon,A., Nicolas,A. and Galibert,F. (2000) POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase , defines a link between DNA replication and the mutagenic bypass repair pathway. Curr. Genet., 38, 178–187.[Web of Science][Medline]
44 Chanet,R., Heude,M., Adjiri,A., Maloisel,L. and Fabre,F. (1996) Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase. Mol. Cell. Biol., 16, 4782–4789.[Abstract]
45 Palladino,F. and Klein,H.L. (1992) Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants. Genetics, 132, 23–37.[Abstract]
46 Liberi,G., Chiolo,I., Pellicioli,A., Lopes,M., Plevani,P., Muzi-Falconi,M. and Foiani,M. (2000) Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and cdk1 activity. EMBO J., 19, 5027–5038.[Web of Science][Medline]
47 Lee,S.K., Johnson,R.E., Yu,S.L., Prakash,L. and Prakash,S. (1999) Requirement of yeast SGS1 and SRS2 genes for replication and transcription. Science, 286, 2339–2342.
48 Gangloff,S., Soustelle,C. and Fabre,F. (2000) Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases. Nature Genet., 25, 192–194.[Web of Science][Medline]
49 Klein,H.L. (2001) Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2 with other DNA repair genes in Saccharomyces cerevisiae. Genetics, 157, 557–565.
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