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Eppley Institute for Research in Cancer and Allied Diseases,2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-68051
Received 28 June 2006/ Returned for modification 20 July 2006/ Accepted 2 October 2006
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since TNR expansions require the addition of DNA, clearly the mechanism must involve a DNA synthetic event. Various models of TNR instability invoke replication and/or repair (28, 39, 42, 60), and each model is by no means mutually exclusive. For proliferating cells, one widely supported model for TNR expansions involves aberrant DNA replication on the lagging strand (18, 25, 36, 38, 49). In support of this idea, DNA synthesis of repetitive elements, such as TNRs, has a high error rate due to polymerase slippage (44). Furthermore, replication forks often stall when synthesizing TNR-containing DNA (43, 52). By this model, a DNA polymerase synthesizing through a TNR-containing region inadvertently provokes formation of a single-stranded region (at either the 3' or 5' end of the DNA being synthesized) that folds into a hairpin or other secondary structure. One version of the model envisions polymerase-mediated strand displacement of the downstream 5' terminus to create a flap (18). Left intact, the flap can fold into a hairpin and be ligated, thus sealing extra TNRs into the newly synthesized strand. The Fen1 flap endonuclease helps remove single-stranded TNR flaps during Okazaki fragment processing, thereby preventing TNR expansions (20, 33, 34). In vivo, the 5' flap model is supported by high TNR expansion rates in yeast deficient in the Fen1-encoding gene RAD27 (13, 34, 54, 57). An alternative view envisions polymerase stalling and 3' terminus slippage (reviewed in reference 60). Slippage at the 3' end allows the polymerase to aberrantly replicate a region of the template more than once. Formation of secondary structure stabilizes this expanded replication intermediate. As noted earlier, polymerase stalling at TNRs has been documented; however there is little direct experimental evidence to support the idea that expansions result from 3' slippage events.
In addition to the important cis factors, such as structure-forming capabilities, trans factors involved in DNA metabolism play important roles in mediating TNR instability as well. In humans, evidence for trans-acting factors include the subset of TNR diseases with a parent-of-origin effect (23, 42, 60). Also, variations in germ line instability between individuals with similar Huntington's disease allele lengths (10, 31) suggest that genetic differences can affect TNR stability quite substantially. As part of the effort to identify relevant trans-acting factors in model organisms, we recently reported Saccharomyces cerevisiae Srs2 as a potent inhibitor of TNR expansions (4). Srs2 has two major functions. It negatively regulates homologous recombination (1, 27, 35, 40, 45, 51, 53, 61), and it positively modulates the RAD6 postreplication repair (PRR) pathway (1, 7, 40, 45, 51, 53, 56). Genetic evidence (4) indicated that the majority of Srs2's inhibitory action for TNR expansions was independent of homologous recombination; therefore, PRR became a focus for investigation.
PRR appears to be well conserved from yeast to humans (6, 46). There are well-established human homologs for most PRR proteins, with the exceptions of Srs2 and Rad5. PRR is a damage tolerance pathway that directs gap filling of aborted DNA replication intermediates arising when a polymerase is unable to proceed past damage on the template strand (6). Although the PRR pathway directs tolerance of both induced and spontaneous forms of DNA damage, evidence indicates that the role PRR proteins play in bypassing spontaneous damage differs from that played in bypassing induced damage (9, 14, 32, 37). For example, although Rad18 is essential for mutagenesis following induced DNA damage, it is only partially required for spontaneous mutagenesis (14). Thus, there appear to be two similar, but distinct, subpathways of PRR: one for bypassing spontaneous lesions and one for bypassing induced lesions.
Trinucleotide repeat mutations are believed to occur spontaneously, and so for ease we will focus only on PRR at spontaneous lesions. For spontaneous damage, evidence suggests that repair is mediated by either a Rad6/Rad18 heterodimer or a Rad5/Rad5 homodimer that assembles on the DNA and directs bypass/tolerance through the modification of PCNA (41). These dimers can direct error-prone, translesion synthesis through polymerase 
(Pol ). Rad6/Rad18-mediated monoubiquitination of PCNA can also direct error-free bypass. This error-free pathway is directed through subsequent polyubiquitination of PCNA mediated by Ubc13, Mms2, and Rad5. Although the mechanism of this pathway is poorly understood, current evidence in Escherichia coli suggests that bypass may occur via a pathway analogous to the template switch pathway mediated by DnaK (17). In support of this, error-free PRR in S. cerevisiae may occur via recombination between partially replicated sister strands in a process that competes with Rad52-mediated homologous recombination (56, 63). Interestingly, error-free PRR has also been linked to completion of replication in response to stresses like hydroxyurea (HU) (5). This evidence suggests that PRR is important for bypassing stalled replication sites as well as damaged templates. Together, the features of PRR and its known relationship to Srs2 made this pathway relevant for study with respect to TNR instability.
In this study, we utilize genetic means to examine the role of PRR in TNR instability. By this approach, we have identified that PRR proteins and PRR-specific PCNA modifications inhibit TNR expansions, and this effect is directed by the action of Srs2. Collectively, this work highlights the importance of a damage bypass pathway in protecting against aberrant DNA replication at TNRs and provides evidence supporting the 3' slippage model of TNR expansions.
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MATERIALS AND METHODS |
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trp1 ura3-52 ade2 ade8 hom3-10 his3-kpn1 met4 met13) (26), was utilized. With the exception of the POL30 lysine mutants, all mutants described in this study are deletions. Most mutants were isogenic derivatives created by targeted deletion techniques and confirmed by PCR and/or phenotypic traits like UV sensitivity. srs2::LEU2 was a random transposon mutant of the wild-type strain isolated in a screen and confirmed as previously described (4). The endogenous POL30 gene was replaced by integrative replacement plasmids, derived from site-directed mutagenesis of the POL30 plasmid pCH1572 (2), to create the pol30-K164R, pol30-K127R, and pol30-K127/164R strains. Integration of the mutant constructs was confirmed by Southern blotting. pol30-K164R and pol30-K164/127R mutants were further verified by UV sensitivity and the loss of UV-induced mutagenesis. The chromosomal integration of TNR-containing plasmids and confirmation of correct single integrants were done as described previously (11). Plasmids. The pBL94 vector was used to construct all TNR-containing plasmids as described previously (50). POL30 replacement plasmids were provided by Polina Shcherbakova, University of Nebraska Medical Center. TRP1-marked pRAD18 was created by blunt end ligation of the 1.1-kb BstUI TRP1 fragment of pRS314 (55) and the 10.6-kb backbone of FspAI-digested YCp11. Plasmid YCp11 (ClaI insert of RAD18 and its endogenous promoter in YCp50) was generously provided by Francis Fabre, Commissariat a l'Energie Atomique. The dinucleotide repeat-containing plasmid, pSH44, was a generous gift of Tom Petes, Duke University.
Genetic assays and molecular analysis of mutated TNR alleles. Expansion and contraction rates were measured by fluctuation analysis as described previously (11, 38, 50). Briefly, for expansions pure TNR tracts were normalized to 25 repeats with randomized C, T, and G deoxyribonucleotides. For example, (CTG)13 tracts have 13 contiguous CTG repeats followed by 36 bp of nonrepeating, randomized C, T, and G deoxyribonucleotides. Normalized TNR tracts were cloned into an S. cerevisiae promoter-reporter construct that allows spacing-sensitive expression of the downstream URA3 reporter. Yeast cells harboring an expansion of four or more repeats do not express URA3 and are identified by their resistance to 5-fluoroorotic acid. For contractions, pure TNR tracts are normalized to 33 repeats, and contractions of 5 or more repeats allow expression of URA3; cells are identified by their ability to grow in the absence of uracil. Mutation rates were calculated by the method of the median (30). Dinucleotide mutation rates were measured as described previously (19). Forward mutation rates for the CAN1 gene were determined by fluctuation analysis with selection for canavanine resistance. Three to five independent clones were tested for each of the above assays to ensure reproducibility. Single-colony PCR analysis of expansions and contractions were done as previously described, and rates were corrected by multiplying the percent bona fide expansions/contractions by the apparent mutation rates obtained by fluctuation analysis (11). All statistical analyses were performed using the t test (two-tailed distribution and two-sample equal variance) in Microsoft Excel, and P values of less than 0.05 were considered statistically significant. Any outliers were determined using the Q-test.
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RESULTS |
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As a model, we chose PRR-mediated bypass of spontaneous lesions (37). This model is the most relevant, since our assay system does not employ DNA damaging agents; therefore TNR expansions and contractions occur spontaneously. As described earlier, PRR of spontaneous DNA damage has been genetically characterized as a three-pathway model, with two error-prone branches (directed by either Rad18 or Rad5) and one error-free branch (mediated by both Rad18 and Rad5). Thus, if PRR helps prevent TNR expansions, then loss of either RAD18 or RAD5 should lead to higher expansion rates. Both (CAG)25 and (CTG)13 expansions were elevated in rad5 (four- to sixfold) and rad18 (eight- to ninefold) (Table 1). Furthermore, this defect in rad18 could be completely rescued by ectopically expressing RAD18 in the mutant cells, but not by an empty vector control, confirming that loss of RAD18 is responsible for the increased level of expansions (Table 1). In addition, for each TNR tract examined, the expansion rates were elevated to a similar extent in both rad18 and rad5, and there was no significant difference between the respective expansion rates (P = 0.25). To further address the relationship between rad18 and rad5, (CTG)13 expansion rates were measured in the rad18 rad5 double mutant. The result was indistinguishable from rad18 and rad5 results (data not shown). While additivity cannot be completely ruled out, this observation is consistent with the idea that Rad5 and Rad18 proteins function in the same pathway to inhibit TNR expansions. Previous work in our lab has illustrated that (CTG)13 tracts expand by +4 to +11 repeats, but (CAG)25 repeats generally have larger expansions (generally +9 to +25 repeats). Since both of these tracts have increased expansion rates in our mutants, these results indicate that PRR is important for inhibiting CAG · CTG repeat expansions over the range of +4 to +25 repeats.
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Spontaneous lesions can be repaired through an error-prone branch or an error-free branch of PRR (37). Error-prone bypass by Pol is directed by Rad18 or Rad5 homodimers, and error-free bypass occurs via a pathway involving both Rad6/Rad18 and Rad5. To distinguish between these possibilities, we measured (CTG)13 expansions in a rev7 strain. A rev7 mutant is devoid of the regulatory subunit of Pol , rendering the polymerase nonfunctional; therefore, this mutant is only capable of error-free bypass. In contrast to rad5 and rad18, we did not observe any significant change in (CTG)13 expansion rates in rev7 (10a). Although Rad30 (Pol 
) is another bypass polymerase that has been implicated in PRR, previous work illustrated that a rad30 strain had little or no detectable change in TNR mutations (12). As a second way to examine the role of error-free PRR, expansions were measured in a ubc13 mutant which is deficient in polyubiquitination of PCNA. The expansion rate of (CAG)25 was elevated sixfold over wild type (Table 1), a phenotype statistically indistinguishable from those of rad18 and rad5. These results, along with the above evidence for PRR inhibition of TNR expansions in rad18 and rad5, suggest that error-free bypass, not error-prone, is important for inhibiting expansions.
To further examine the role of PRR inhibition of TNR expansions, PCNA modifications were examined genetically. Recent advances in the PRR field have illustrated that modifications of PCNA are vital for dictating the different outcomes of PRR. Two independent modifications of PCNA are relevant for PRR, sumoylation and ubiquitination. K164 of PCNA is the predominant site of PCNA ubiquitination and sumoylation. Sumoylation at this site enhances Srs2's interaction with PCNA and presumably enhances Srs2's recruitment to stalled replication forks (40, 45). Notably, sumoylation is not essential for PRR (40), and sumoylated PCNA has not been detected in human cells. PCNA becomes monoubiquitinated (by Rad6/Rad18) and/or polyubiquitinated (by Rad5 and other proteins) at K164 to direct the various outcomes of the PRR pathway (22). A second residue, K127, can also be sumoylated. This sumoylation appears to be secondary to K164 sumoylation and is generally not believed to be crucial for PRR (40, 45).
To test whether these modification sites are important for TNR instability, expansion rates were measured in pol30-K127R, pol30-K164R, and pol30-K127/164R mutants. Mutation of these lysine residues to arginine abolishes their ability to be sumoylated and ubiquitinated. Results in Table 1 show that elevated levels of (CTG)13 and (CAG)25 expansions were observed in strains harboring pol30-K164R (up to 45-fold) and pol30-K127/K164R (up to 9-fold), but substantial elevations were not observed for pol30-K127R. Modifications at K127 are not crucial for inhibiting TNR expansions, whereas modifications at K164 help to inhibit expansions. These results support the idea that PRR inhibits TNR expansions, since PCNA mono- and polyubiquitination at K164 is required for PRR. Interestingly, there is a fivefold difference between the TNR expansion rates of pol30-K164R and both pol30-K127/164R and srs2. Previous work suggested that the higher spontaneous mutation rate of pol30-K164R compared to pol30-K127/164R is dependent on Pol (58). This does not appear to be the case for TNR expansions, however, because expansion rates of pol30-K164R and the double mutant pol30-K164R rev7 are indistinguishable from one another (data not shown). Although there is no clear explanation for the higher rate of expansions in pol30-K164R compared to other PRR mutants, a similar situation was observed for sensitivities to DNA damaging agents, where pol30-K164R is more sensitive than either srs2 or pol30-K127/164R (40).
To address the relationship between PCNA modifications and Srs2, comparisons were made between (CTG)13 expansion rates of srs2, pol30-K164R, and the double mutant srs2 pol30-K164R. The comparisons are consistent with an epistatic relationship between the two mutants (Table 1). Since the double mutant is not significantly different from srs2 (11-fold versus 13-fold, respectively), it implies Srs2 acts upstream of K164 modification to inhibit TNR expansions.
To gain insight into the types of TNR mutations occurring in PRR mutants, expansion sizes were measured. Previous reports with srs2 illustrated that (CTG)13 tracts usually expand by 5 to 10 repeats with a bell-shaped spectrum, which is similar to a wild-type spectrum (+4 to 11 repeats) (4). Likewise, expansions from (CTG)13 starting tracts from rad18 and pol30-K164R were in the range of +4 to 11 repeats (Fig. 1). Comparisons between the expansion spectra of the wild type, srs2 (4), rad18, and pol30-K164R demonstrate that the expansion ranges overlap, suggesting the same types of expansion events are occurring and no unique expansion events have emerged. Thus, as with srs2, the sheer number of expansions accounts for the increased expansion rate observed in PRR mutants.
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Synergistic interaction between Rad27 and PRR for TNR expansions. Rad27 is a well-characterized trans-acting factor for inhibiting TNR expansions (13, 20, 33, 34, 54, 57). These findings are in excellent agreement with models for TNR expansions resulting from 5' flaps which then fold into hairpin structures (18). An alternative expansion model posits that 3' strand slippage occurs if DNA polymerases stall or pause in a TNR tract (reviewed in reference 60). By this model, the slipped strand folds into a hairpin with a 3' terminus in the duplex region, which can then be extended by a polymerase. To date, however, data to directly support expansions from 3' slippage have not been available. PRR processing is predicted to be recruited to an intermediate containing a 3' end where replication has been stalled (59); therefore, our data with PRR mutants may indicate expansions from 3' slippage. If so, inhibition of TNR expansions by Rad27 and PRR would be exclusive of one another and should be genetically distinct. A model to further clarify this hypothesis is shown in Fig. 2.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Based on these findings, we propose a model for spontaneous PRR of TNRs (Fig. 3) that is based largely on the spontaneous lesion bypass pathway described by Jinks-Robertson and colleagues (37). We propose that DNA synthesis through TNRs results in hairpin formation and/or polymerase stalling, and this signals recruitment of Srs2 and the PRR machinery. Action by Srs2 and PRR helps to surpass the replicative stress (i.e., fork stalling and/or hairpin formation) through faithful maintenance of the TNR tract in a manner dependent on mono- and polyubiquitination of PCNA. Since the mechanism of error-free PRR is still poorly understood, the precise mechanism that allows error-free synthesis of the TNR tract remains enigmatic. This model is supported by several pieces of evidence. First, in this study we have shown that TNR expansions are elevated in mutants that block each step shown in Fig. 2. Second, it is well established that replication forks often stall at TNR tracts (43, 52). However, it is unknown whether stalling is caused by the TNR tract alone or following hairpin formation. Third, the HU sensitivity of PRR mutants suggests that PRR proteins are recruited to help resolve stalled replication forks (5), and so it is reasonable to suggest that PRR helps relieve stress associated with DNA synthesis through TNR tracts. What happens to a TNR hairpin in the absence of PRR is an open question. Either the hairpin persists and leads directly to an expansion, or some alternative processing event actively promotes expansion in an error-prone manner. Additional work will be necessary to distinguish between these two possibilities.
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A synergistic interaction between PRR and Rad27 suggests that these pathways are not completely independent and each may compensate for the loss of the other. One possible explanation for this result is that loss of either Rad27 or PRR increases the formation of TNR structures that are substrates for the remaining pathway. For example, in the absence of Rad27, 5' flaps can reequilibrate into various alternative structures (21, 62) which may be substrates for PRR. One possible intermediate would arise if the 5' flap reannealed to the template and displaced the upstream Okazaki fragment (62), resulting in a 3' TNR slip-out. This seems like a reasonable substrate that may recruit PRR. Alternatively, in the absence of PRR, TNR loop-outs at the 3' end would persist and might also reequilibrate with downstream Okazaki fragments to generate 5' flap substrates for Rad27.
In addition to directing PRR to the triplet repeat tract, Srs2 may play a second role by unwinding the putative hairpin intermediate. This possibility is supported by biochemical experiments showing that purified Srs2 is unusually active at unwinding oligonucleotide substrates containing TNR repeats (3). If so, one might predict that sumoylation of PCNA by Siz1 might help recruit Srs2 to the hairpin in vivo, based on studies from the antirecombination properties of Srs2 (40, 45). Interestingly, a siz1 mutation did not appreciably affect expansions (Table 2), suggesting that PCNA sumoylation is not essential for inhibiting CAG · CTG expansions in this system. If Srs2 does have a second role to unwind the hairpin intermediate, it does not seem to require recruitment by PCNA sumoylated at residue K164.
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ACKNOWLEDGMENTS |
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We thank Frances Fabre for providing YCp11, Wei Xiao for the DBY747 strains, Tom Petes for pSH44, Polina Shcherbakova for POL30 replacement plasmids, and Helle Ulrich for biochemical confirmation of PCNA sumoylation.
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FOOTNOTES |
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Published ahead of print on 23 October 2006.
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