Saccharomyces cerevisiae Srs2 DNA Helicase Selectively Blocks Expansions of Trinucleotide Repeats

Trinucleotide repeats (TNRs) undergo frequent mutations in familiesafflicted with certain neurodegenerative disorders and in modelorganisms. TNR instability is modulated both by the repeat tractitself and by cellular proteins. Here we identified the Saccharomycescerevisiae DNA helicase Srs2 as a potent and selective inhibitorof expansions. srs2 mutants had up to 40-fold increased expansionrates of CTG, CAG, and CGG repeats. The expansion phenotypewas specific, as mutation rates at dinucleotide repeats, atunique sequences, or for TNR contractions in srs2 mutants werenot altered. Srs2 is known to suppress inappropriate geneticrecombination; however, the TNR expansion phenotype of srs2mutants was largely independent of RAD51 and RAD52. Instead,Srs2 mainly functioned with DNA polymerase delta to block expansions.The helicase activity of Srs2 was important, because a pointmutant lacking ATPase function was defective in blocking expansions.Purified Srs2 was substantially better than bacterial UvrD helicaseat in vitro unwinding of a DNA substrate that mimicked a TNRhairpin. Disruption of the related helicase gene SGS1 did notlead to excess expansions, nor did wild-type SGS1 suppress theexpansion phenotype of an srs2 strain. We conclude that Srs2selectively blocks triplet repeat expansions through its helicaseactivity and primarily in conjunction with polymerase delta.

INTRODUCTION

Top

Introduction

Trinucleotide repeats (TNRs) undergo frequent expansions infamilies afflicted with Huntington's disease, fragile X syndrome,and at least 13 other neurodegenerative diseases (7, 8, 35).In addition to this biomedical relevance, the unusual geneticmechanisms underlying TNR instability have sparked substantialscientific interest. Studies in the field of human geneticsand with model organisms indicate that the TNR DNA itself playsa major role in its own mutability (7, 8, 17, 35, 45). For example,the risk of expansion is closely tied to cis-acting features,such as the sequence and length of the TNR tract, and whetherthe repeat is perfect or imperfect. While these "rules" canusually predict expansion risk, there are clear exceptions.For example, the risk of an expansion can vary greatly dependingon whether the gene is inherited maternally or paternally (35,45). In addition, genetic background and other factors havebeen suggested to explain, for example, the variations in germline instability among individuals with similar allele lengthsat the Huntington's disease locus (22).

These observations provide some of the evidence suggesting thatcellular proteins modulate the likelihood of TNR instability.What proteins might be involved? Important clues come from modelsof expansion (7, 12, 17, 45). While expansions result from morethan one genetic mechanism, a central feature of essentiallyall expansion models is that single-stranded TNR sequences foldinto aberrant DNA structures, usually hairpins (11), which arecrucial intermediates in the mutation process (12, 17, 45).If a hairpin cannot be prevented from forming or is not removedquickly enough, an expansion will ensue. Thus, hairpins arethought to be direct precursors of expansions. Proteins thateither prevent hairpin formation or accelerate hairpin removalwould help reduce expansion rates. One well-characterized proteinthat helps block hairpin formation is the flap endonucleaseFEN-1 (24), which removes some single-stranded TNR flaps (10,24, 25, 40, 43, 44). However, it is likely that other proteinsalso help avoid expansions.

In this study, the Srs2 DNA helicase was identified in a geneticscreen for inhibitors of expansion in Saccharomyces cerevisiae.Characterization of srs2 mutants indicates that the helicaseis a potent and selective inhibitor of TNR expansions.

MATERIALS AND METHODS

Top

Materials and Methods

Strains. Most of the S. cerevisiae strains used were derived from MW3317-21A(18) (MAT ${alpha}$ ${Delta}$ trp1 ura3-52 ade2 ${Delta}$ ade8 hom3-10 his3-Kpn1 met4 met13).Isogenic derivatives containing disruptions of msh2, rad27,rad52, or rad51 were constructed by standard techniques (26,39) and confirmed by Southern blotting and phenotypic analysis.The strain harboring the srs2::TRP1 mutation, HKY723-5D (MATahpr5::TRP1 leu2-3,112 his3-11, 15 ade2-2 ura3-1 trp1-1 can1-100RAD5⁺), was kindly provided by Hannah Klein, New York UniversitySchool of Medicine. A strain harboring sgs1::kanMX4 was obtainedfrom Open Biosystems. BL 492, a pol32 ${Delta}$ derivative of BL490 (MATaleu2 ${Delta}$ 1 trp ${Delta}$ 63 ura3-52 his3-200) was a generous gift from SergeiMirkin (University of Illinois, Chicago). The chromosomal integrationof TNR-containing plasmids and confirmation of correct singleintegrants were done as described previously (33, 37).

Plasmids. All triplet repeat-containing plasmids were constructed withthe pBL94 vector as described previously (38). Plasmids pHK209(BamHI-SalI insert of SRS2 in YCp50), pHK282 (EcoRI-SalI insertof srs2K41A in pRS314) and pFP56 (srs2::TRP1) were generousgifts from Hannah Klein. To create low-copy-number pSRS2 plasmids,the SRS2 coding fragment was recovered as a BamHI-SalI fragmentfrom pHK209 and subcloned into pRS314 (marked with TRP1) orpRS315 (marked with LEU2) (42). When low-copy-number pSRS2 wasused for complementation studies of the srs2::LEU2 and srs2::TRP1alleles, the appropriate pSRS2 plasmid was always added to thestrain already containing the triplet repeat reporter. The high-copy-numberpSRS2 plasmid used for overexpression studies was created bytransfer of the SRS2 gene as an EcoRI-SalI fragment from pHK209into pRS424 (6). The reporter plasmid pSH44, containing 16.5repeats of the dinucleotide repeat GT (14), was kindly providedby Tom Petes, University of North Carolina. The 2µm plasmidYEplac195-SGS1 was a generous gift from Alan Morgan, Universityof Liverpool. For this study, the SGS1 gene was moved as a SalIfragment into the corresponding site of the high-copy-numberplasmid pRS424.

Genetic assays and molecular analysis of mutated TNR alleles. Expansion and contraction rates were measured by fluctuationanalysis as described previously (33, 37). Briefly, TNR tractswere cloned into a yeast promoter-reporter to create spacing-sensitiveexpression of a downstream URA3 gene. Tracts containing up to25 TNRs or a subset of repeats plus randomized sequence DNAequivalent to 25 repeats were used to score expansions of +5repeats or more (33, 37). To measure contraction rates, theassay is performed with longer starting tracts (here we used25 repeats plus 24 random-order nucleotides, equivalent to eightrepeats), referred to as CTG₂₅₊₈ (37) Contractions of –5or more repeats are identified as Ura⁺ colonies. Dinucleotiderepeat mutation rates were measured as described previously(14). Forward mutation rates for the CAN1 gene were determinedby selection for canavanine resistance. Three to five independentclones were tested for each of the above assays to ensure reproducibility.Single-colony PCR analysis of TNR expansions and contractionswas performed by a published method (33). The percent bona fideexpansions or contractions, determined by PCR analysis, wasmultiplied by the apparent expansion and contraction rates derivedfrom fluctuation analysis. All rates reported here reflect thiscorrection factor.

Western blot analysis. The expression of Srs2 was analyzed by Western blotting. Briefly,10⁷ cells/ml from a 5-ml overnight culture were harvested andlysed in sample buffer with glass beads; 10 µl of thislysate (containing 5 µg of total protein) was loaded ontoa sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) 7.5% polyacrylamide gel and separated by electrophoresis,the proteins were transferred to a nitrocellulose membrane (Amersham),and the membrane was blocked overnight at 4°C in Odysseyblocking buffer (Li-Cor). Immunodetection was done with rabbitanti-Srs2 antiserum (from Patrick Sung, Yale University) orwith anti-Rfc3 antiserum (from Peter Burgers, Washington University),followed by probing with Alexa Fluor 680 goat anti-rabbit immunoglobulinG (Molecular Probes) and fluorescence detection and quantitationwith the Odyssey system, per the manufacturer's protocol.

Helicase substrate analysis and unwinding assays. End labeling with ³²P by T4 polynucleotide kinase (New EnglandBiolabs) was performed according to the manufacturer's protocol.Oligonucleotide strands (one radiolabeled) were mixed at equimolarconcentrations with the unlabeled complement, heated at 95°Cfor 5 min, and then slowly cooled to room temperature. The locationof the radiolabel on either the longer or shorter strand isstipulated in the legends to Fig. 3 and 4. Mung bean nuclease(New England Biolabs) digestions (total volume, 20 µl)were performed with 1 unit of enzyme in the manufacturer's reactionbuffer at 37°C for 40 min. Reactions were quenched by additionof SDS to 0.01% final concentration, followed by ethanol precipitation,resuspension in formamide loading buffer, and analysis on a15% sequencing gel.

View larger version (15K):
[in this window]
[in a new window]
FIG. 3. Srs2 unwinds CTG repeat-containing double-stranded DNA. (A) Schematic diagram of putative Srs2 action on a CTG hairpin in vivo. In the diagram, a newly synthesized CTG tract has folded onto itself to form a hairpin (the template strand has been omitted for clarity). Srs2 (diamond) loads onto the 3' DNA end and unwinds the structure. The single-stranded product might then reanneal properly to the template strand or might be subject to nuclease digestion, followed by resynthesis of the tract. Either outcome would help eliminate hairpins and thereby reduce the appearance of expansions. (B) Partial DNA duplexes containing CTG repeats can be tested in vitro as potential Srs2 helicase substrates. Srs2 action is envisioned to work as in A. The products of unwinding are single-stranded DNAs which can be readily assayed by gel electrophoresis. (C) Predicted structure of the partially double-stranded substrate. The 3' tail of the longer strand provides a loading site for the helicase. The duplex contains five CTG repeats to mimic a TNR hairpin. The nine complementary base pairs add thermodynamic stability to the duplex and help ensure that the CTG repeats align as predicted. The arrow shows the predicted junction between single- and double-stranded DNA; 24 nucleotides of the bottom strand should be protected from mung bean nuclease, an enzyme that cleaves single-stranded DNA but not double-stranded regions. (D) Nuclease analysis of DNA structure. The annealed substrate, 5'-end labeled on the longer strand with ³²P, was incubated with mung bean nuclease. The products were analyzed on a denaturing 15% polyacrylamide gel. Lanes 1, 2, and 3 contain molecular size markers of 34, 24, and 9 nucleotides, respectively. Lane 4 contains the undigested helicase substrate, and lane 5 contains the product after nuclease digestion. (E) Srs2 helicase activity. The DNA substrate, 5'-end labeled on the shorter strand with ³²P, was incubated under standard helicase assay conditions (Materials and Methods) with 50 nM Srs2 for the indicated times. ${Delta}$ , heat-denatured control.

View larger version (29K):
[in this window]
[in a new window]
FIG. 4. Srs2 unwinds a TNR substrate substantially faster than does UvrD. DNA substrates radiolabeled on the shorter strand were incubated with either Srs2 (35 nM) or UvrD (200 nM) under standard conditions (19, 30) for the indicated times, and the reaction products were separated by PAGE and visualized by phosphorimaging. Both DNA substrates contained a 14-nucleotide-long 3' tail, which is usable by either Srs2 or UvrD. (A) Schematic diagram of the (CTG)₁₀-containing substrate. Along with the 9 bp of nonrepeating DNA (identical to Fig. 3C), the duplex region is 39 bp in total length. (B) Control substrate (19) without triplet repeats. The duplex region measures 40 bp.

For helicase assays, the radiolabeled partial duplex DNA substratewas incubated with 25 to 35 nM purified Srs2 for 0 to 15 minunder standard helicase assay conditions; 30°C in the presenceof 300 nM (nucleotides) DNA substrate, 25 mM Tris-Cl (pH 7.5),2.5 mM MgCl₂, 1 mM dithiothreitol, 100 µg of bovine serumalbumin per ml, and 2 mM ATP (19). UvrD was used at 200 nM for0 to 20 min as follows (30); the reaction mixture contained25 mM Tris-Cl (pH 7.5), 3 mM MgCl₂, 20 mM NaCl, 50 mM 2-mercaptoethanol,50 µg of bovine serum albumin per ml, and 10 nM DNA substrate.UvrD was diluted in helicase II storage buffer and then incubatedfor 5 min at 37°C with the reaction mix lacking ATP. Theunwinding reaction was initiated by addition of ATP to a finalconcentration of 3 mM. For all helicase reactions, the DNA productswere subsequently analyzed by nondenaturing 15% PAGE.

RESULTS

Top

Results

Identification of SRS2 as a gene affecting triplet repeat expansions. To find candidate genes that help protect the cell from expansions,random transposon mutants of S. cerevisiae were screened forincreased expansions of a (CTG)₁₃ reporter (4). A CTG sequencewas used because CTG•CAG tracts are unstable in severalhuman genetic diseases. The length of 13 is relevant in S. cerevisiaebecause it falls close to a crucial threshold length (37) atwhich expansions become increasingly more frequent. We reasonedthat yeast mutations that alter (CTG)₁₃ expansion rates mightbe specific for TNRs, since thresholds are unique to TNRs. Thresholdsare well known in human TNR instability, where lengths of ${approx}$ 35repeats are associated with increased risk of expansion (35).

DNA sequencing (4) identified srs2::LEU2 as a disrupted genethat gave increased expansion rates. Additional genetic assayswere performed to verify the srs2 mutation. Both the srs2::LEU2mutant and an srs2::TRP1 mutant showed moderate UV sensitivity,consistent with published results (1). Nonhomologous end joining,measured by a plasmid repair assay, was reduced 2.5-fold inthe srs2::LEU2 background, in good agreement with publishedvalues (13). Identical expansion phenotypes were observed withboth the srs2::LEU2 and the srs2::TRP1 strains, and a low-copy-numbernumber pSRS2 plasmid reversed the hyperexpansion phenotype ofthe srs2::LEU2 mutation (see below). Together with the sequencingdata, these results verified SRS2 as the mutated gene.

The hyperexpansion phenotype of the srs2 mutant was surprisingbecause neither spontaneous nor damage-induced mutagenesis,measured as forward mutations at CAN1, is increased in srs2strains (2). To evaluate the hyperexpansion phenotype more thoroughly,quantitative mutation rates were determined. Expansions of the(CTG)₁₃ tract (37) were elevated 40- to 44-fold over that inthe wild type for our srs2::LEU2 allele and for the srs2::TRP1strain obtained from another laboratory (Table 1). The wild-typeSRS2 gene on a low-copy-number plasmid (pSRS2) reduced the expansionrate to wild-type levels. The expansion sizes of CTG tractsin srs2::LEU2 were also measured (33). Starting from an initial(CTG)₁₃ tract, the expanded alleles ranged from +5 to +10 repeats,with a median of +8, similar to expansions in the wild-typestrain (Fig. 1). As the two mutational spectra overlap, we concludethat the number of expansions, not their size, accounts forthe dramatic increase in the expansion rate of the srs2 strain.There was a single case of a large expansion (+31 repeats) inthe srs2::LEU2 mutant (Fig. 1). However 98% of CTG expansionsin the srs2 strain were less than twice the original tract size,consistent with a replicational model of TNR repeat instability(12).

View this table:
[in this window]
[in a new window]
TABLE 1. Mutation rates in srs2 mutant strains^a

View larger version (14K):
[in this window]
[in a new window]
FIG. 1. (CTG)₁₃ expansion sizes in wild-type and srs2::LEU2 strains. A subset of the 5-fluoroorotic acid-resistant colonies obtained from fluctuation analysis were subjected to single-colony PCR analysis and sizing on sequencing gels to an accuracy of ±1 to 2 repeats (33). The x axis denotes the number of repeats added to the original tract of 13 repeats.

Hyperexpansion phenotype of an srs2 mutant is mainly associated with DNA polymerase delta, not with genetic recombination. Genetic and biochemical studies (1, 3, 19, 47) indicate thatSrs2 functions as an antirecombinase, and many srs2 phenotypesare linked to recombination (16). Therefore, it was importantto know whether the hyperexpansion phenotype of srs2 was alsoa result of hyperrecombination. If so, expansion rates shouldbe reduced to near wild-type levels when recombination is inactivated.In contrast to this prediction, the results in Table 1 showthat double mutants (rad51 srs2 and rad52 srs2) still retainedmuch of the instability (11- to 33-fold) seen in an srs2 singlemutant (40- to 44-fold). Thus, loss of recombination reducedthe srs2 effect on CTG expansions by only 1.3- to 4-fold. Whilea portion of the srs2 expansion phenotype might be due to recombinationalmisprocessing of TNRs, some other process is responsible forthe majority of the phenotype. Expansion rates were also unaffectedwhen either rad51 or rad52 was mutated singly (Table 1), arguingagainst unequal sister chromatid exchange (9) as a mechanismof expansion.

An alternative possibility is that Srs2 functions with DNA polymerasedelta to block expansions. This idea is based on the findingthat Srs2 interacts in two-hybrid experiments with the Pol32subunit of polymerase delta (15). If Srs2 and polymerase deltaare both needed for inhibiting TNR expansions, then a mutantlacking POL32 should show a high expansion rate. A (CTG)₁₃ tractwas 29-fold more unstable in a pol32 ${Delta}$ strain than its wild-typecounterpart (Table 1). Similarly, the repeat tract was 20-folddestabilized in an srs2 pol32 double mutant. This epistasisanalysis is consistent with the idea that the majority of expansionsprevented by Srs2 occur in conjunction with Pol32, and hencepolymerase delta. Statistical analysis by Student's t test showedno significant differences between the expansion rates for srs2compared to pol32 ${Delta}$ (P = 0.08) or for pol32 ${Delta}$ compared to srs2 pol32 ${Delta}$ (P = 0.14); however, there was a significant difference betweensrs2 and srs2 pol32 ${Delta}$ (P = 0.04).

The epistasis relationship between Srs2 and polymerase deltamay be somewhat complex. To examine this idea further, we testedthe effect of Srs2 overexpression. If a direct physical interactionbetween Srs2 and Pol32 is important for preventing expansions(for example, to recruit Srs2 to a replication fork), overexpressingSrs2 in a pol32 ${Delta}$ background should not alleviate the hyperexpansionphenotype. In contrast to the prediction, we found that theexpansion rate for Srs2 overexpression in a pol32 ${Delta}$ strain wasindistinguishable from wild-type levels (Table 1). Immunoblotting(Fig. 2B) showed that Srs2 levels were elevated three- to fourfoldwhen cells harbored SRS2 on a multicopy plasmid. We concludethat Srs2, when overexpressed, does not require physical interactionwith Pol32 to block expansions. Caution is warranted when interpretingthis result, however, because the interaction may be more importantwhen Srs2 is present at normal levels.

View larger version (32K):
[in this window]
[in a new window]
FIG. 2. Immunoblot analysis of Srs2 expression. Whole-cell extracts were separated by SDS-7.5% PAGE, transferred to a nitrocellulose filter, blotted with polyclonal Srs2 antiserum, and detected by fluorescence. (A) Lane 1, purified Srs2. Lane 2, extract from srs2::LEU2 cells. Lane 3, extract from srs2::LEU2 cells containing low-copy-number pSRS2 plasmid. Lane 4, extract from srs2::LEU2 cells containing the low-copy-number mutant plasmid psrs2K41A. Bottom strip, loading control immunoblot for Rfc3. Similar results were seen in two other repetitions of this experiment. (B) Lane 1, extract from wild-type cells. Lane 2, extract from wild-type cells containing a high-copy-number pSRS2 plasmid. Lane 3, extract from pol32 ${Delta}$ cells. Lane 4, extract from pol32 ${Delta}$ cells with a high-copy-number pSRS2 plasmid. A similar result was seen in a repetition of this experiment.

Srs2 selectively blocks TNR expansions. Expansion rates were also examined for a longer TNR, (CTG)₂₅.In a previous study (37), we showed that (CTG)₂₅ is above theapparent threshold length of 13 repeats in S. cerevisiae. Asa reference, wild-type cells undergo expansions of (CTG)₂₅ 70-foldmore often than for (CTG)₁₃ (37). It was therefore of interestto know whether Srs2 can block expansions of a TNR above thethreshold, where expansions occur at a relatively high rateeven in the wild type. We found that the absence of Srs2 furtherincreased instability for (CTG)₂₅. Expansion rates (Table 1)were approximately fivefold higher in the srs2::LEU2 and srs2::TRP1mutants compared to the wild type, and the low-copy-number pSRS2plasmid complemented the srs2::LEU2 mutation. Thus, Srs2 stillprovides protection against expansions for longer CTG tracts.Two other TNRs capable of forming hairpins, (CAG)₂₅ and (CGG)₂₅,showed 3.4- to 4.6-fold enhancement of expansion rates in ansrs2 mutant, respectively (Table 1), suggesting that Srs2 suppressesexpansions for at least three different TNR sequences. In contrast,srs2 had no effect on the non-hairpin-forming and geneticallystable repeat (CTA)₂₅ (see supplemental material at http://www.unmc.edu/Eppley/publications/chart_lah3.html).Similarly, there was no detectable increase in mutation ratesfor poly(GT) dinucleotide repeats (Table 1) or for forward mutationsthat inactivate the CAN1 gene (Table 1). Together, these resultsindicate a high degree of specificity for Srs2 on TNR sequencescapable of hairpin formation.

TNRs also undergo contractions, in which repeating units aredeleted. It is thought that contractions also occur via a hairpin-dependentmechanism, although a hairpin on the template strand is predictedto lead to contractions, not on the daughter strand, as forexpansions. There was no significant effect of srs2 on contractionsof a (CTG)₂₅ reporter (37) (Table 1). Contractions of a shortertract, (CTG)₁₅ (37), were also essentially unaffected by thesrs2 mutation (3.9 x 10^–6 ± 1.2 x 10^–6 versus2.4 x 10^–6 ± 0.6 x 10^–6 for the wild type).These results suggest that Srs2 acts primarily on the daughterDNA strand, not the template strand, to block triplet repeatinstability. Alternatively, the mechanisms of expansion andcontraction might be somewhat different and therefore differentiallysensitive to the loss of Srs2, even though hairpins likely mediateboth types of mutation.

Srs2 helicase activity is important for preventing expansions. Disease-causing TNRs form hairpins (11) that are likely precursorsof expansions (12, 17, 45). Since Srs2 has helicase activity(2, 16, 19, 47), we hypothesized that Srs2 helps prevent expansionsby unwinding hairpins. If so, the ATPase activity of Srs2 shouldbe important for blocking expansions in vivo. We found thatan srs2 point mutant lacking ATPase function, srs2K41A (19),gave a (CTG)₁₃ expansion rate within about twofold of that ofthe null allele (Table 1). The expression levels of the wild-typeand Srs2K41A proteins were similar (Fig. 2A), so the expansiondefects in the K41A mutant cannot be attributed to low expression.

If Srs2 acts in vivo to unwind triplet repeat hairpins and preventexpansion, then perhaps the activity can be mimicked in vitrowith double-stranded DNA containing CTG repeats within the duplexregion (Fig. 3A and B). A DNA substrate was designed to testthis idea (Fig. 3C), based on the following rationale. A single-stranded(CTG)₁₃ repeat tract (the presumed intermediate during expansionin vivo) can fold into a hairpin with maximally five to sixrepeats on one side of the stem, a short loop, and then fiveto six repeats on the other side of the stem. Thus, a helicasesubstrate with five CTG repeats (Fig. 3C) is reasonable. Controlexperiments indicated that the molecule adopted the predictedstructure (Fig. 3D). Srs2 helicase readily unwound the DNA substrate(Fig. 3E) in an ATP-dependent manner (data not shown). The factthat Srs2 was able to unwind duplex DNA containing CTG repeatssupports the idea that Srs2 prevents expansions by unwindingTNR hairpins.

To address the specificity of helicase action, we compared Srs2to UvrD from Escherichia coli. UvrD protein, also called DNAhelicase II, was chosen for comparison because it has been wellstudied (29, 31, 32) and has the same 3'-to-5' polarity (28)as Srs2. If Srs2 is especially active at unwinding TNR-containingsubstrates, then perhaps there would be a difference in helicaseactivity compared to UvrD. Figure 4A demonstrates the activityof both enzymes on a (CTG)₁₀ partial duplex. Srs2 completelyunwound the substrate by the 10-min time point, approximatelytwice the time required to unwind (CTG)₅ (compare Fig. 4A and3E). In contrast, bacterial UvrD proceeded slowly through the(CTG)₁₀ substrate (Fig. 4A), requiring 20 min for full unwindingdespite being assayed at a higher enzyme concentration (200nM for UvrD versus 35 nM for Srs2). Both enzymes were activeon a control substrate without repeats (Fig. 4B). The differencein UvrD's activity cannot be attributed to different duplexlengths, since the total duplex region in the two substrateswas 39 and 40 bp long. The findings in Fig. 4 suggest that eitherthe sequence or the structure or both of the (CTG)₁₀ substratepresents a barrier to unwinding that can be overcome by Srs2faster than by UvrD, another 3'-to-5' helicase.

We also asked whether a related yeast helicase, Sgs1, couldsubstitute for Srs2 in vivo. Sgs1 is a member of the RecQ familyof DNA helicases with 3'-5' polarity (16). SRS2 and SGS1 mutantsshare several phenotypes, but single mutants also show differences(16). Although sgs1 mutants show increased gross chromosomalrearrangements (34), an sgs1::kanMX4 strain in our assay gavea low expansion rate that was very close to the wild-type value(Table 1). Thus, sgs1 and srs2 mutants have different phenotypesfor TNRs. In some circumstances, SGS1 in high copy number cansuppress srs2 phenotypes (27). However, a multicopy plasmidcontaining SGS1 (27) failed to suppress the expansion defectconferred by srs2::LEU2 (Table 1). Our results that SRS2 andSGS1 behave differently at TNRs are in general agreement withthose of White et al. (48), who found that an sgs1 mutationcaused stabilization (not destabilization) of CGG arrays. Theseresults suggest that Srs2 plays a unique role in stabilizingtriplet repeat tracts that cannot be replaced by the functionallysimilar protein Sgs1.

DISCUSSION

Top

Discussion

We identified srs2 in a blind screen for mutations that resultin high expansion rates. Subsequent analysis showed that CTG,CAG, and CGG repeats are destabilized in srs2 mutants. The srs2mutator phenotype is specific for TNR expansions, as there wasno detectable increase in srs2 backgrounds for TNR contractions,dinucleotide repeat alterations, or mutations in unique DNAsequences. Epistasis analysis indicated that the srs2 expansionphenotype was mostly associated with DNA polymerase delta, withonly a fraction of the expansion rate in srs2 mutants attributableto recombination. Expansion sizes in an srs2 mutant were alsoconsistent with a replicational source. The helicase activityof Srs2 is important for preventing expansions in vivo (althoughthe related helicase Sgs1 cannot compensate for loss of Srs2),and purified Srs2 is substantially faster than bacterial UvrDhelicase at unwinding a DNA substrate that mimics a TNR hairpin.Together these results indicate a novel, specific, and directrole for Srs2 in preventing TNR expansions.

What molecular features might account for the selective inhibitionof TNR expansions by Srs2? We suggest that Srs2 acts in vivoto unwind most TNR hairpins before they become fixed as expansions.The unwound hairpin might either reanneal in register with thetemplate strand or become a substrate for nuclease cleavageand thereby remove the excess DNA prior to completion of theexpansion process. This idea helps explain why srs2 strainsare mutators only at TNRs, which are unique among microsatellitesin using a hairpin-based mechanism to expand in large increments.Other repeating elements such as dinucleotides change lengthprimarily one or two repeats at a time, due to replication slippage(41), and thus the small loop-out intermediates would not presenta target for Srs2 helicase action. To explain the stabilizationof TNR expansions but not contractions, it is possible thatSrs2 may not have access to hairpins on the template strandor that loading of the helicase may be precluded. If true, contractionscould occur regardless of Srs2 status.

The final issue is the TNR length dependence of the srs2 phenotype.While both 13-repeat and 25-repeat tracts are protected by Srs2,a greater protective effect is seen for the shorter allele.Perhaps Srs2 helicase is more active on shorter hairpins, butlonger hairpins are not unwound as readily. Helicase assaysshowed that longer times are required for Srs2 unwinding of10-repeat versus 5-repeat substrates. Alternatively, the protectivefunction of Srs2 may be reduced for the (CTG)₂₅ tract becauseit is already relatively unstable. These possibilities willbe addressed in future work.

Our results also suggest that Srs2 helicase prevents expansionsprimarily in conjunction with DNA polymerase delta, since thesrs2K41A mutant, the pol32 ${Delta}$ mutant, and the srs2 pol32 ${Delta}$ doublemutant all gave high (CTG)₁₃ expansion rates within twofoldof the srs2 value. Strains bearing the srs2 or pol32 ${Delta}$ mutationalso showed a similar dependence on repeat tract length; for(CTG)₂₅, a smaller increase in expansion phenotype was seenfor srs2 (Table 1) and no significant effect was seen in pol32 ${Delta}$ strains (36). Polymerase delta is essential for replication,but it also acts during recombination and in some repair pathways(5). Recombination is not a major source of TNR instabilityin our system, as the loss of RAD51 or RAD52 functions in srs2backgrounds reduced the expansion rate by 3.5-fold or less comparedto an srs2 single mutant. Srs2 and polymerase delta must workprimarily through replication and/or repair to inhibit expansions.A role for Srs2 in DNA replication has been suggested previously(21), and expansion sizes in an srs2 mutant are consistent withreplication-mediated events (12). We are not aware of any reportsin the literature testing whether repeat replication is compromisedin srs2 mutants, but this would be a very interesting questionto address.

Several models consistent with our epistasis results can beenvisioned for inhibiting expansions. The first is a directprotein-protein interaction of Srs2 and polymerase delta. Forexample, the polymerase might stall when it synthesizes a TNRtract that subsequently folds into a hairpin and then recruitSrs2 for unwinding. A putative Srs2-polymerase delta complexmight help explain the circa twofold reduction in expansionrates for srs2K41A compared to the srs2 null mutant. This differenceallows for the possibility that there is an ATPase-independentcomponent for inhibiting expansions that can be conferred bythe point mutant protein (perhaps via protein-protein contacts).Our viewpoint is that the large majority (17-fold) of the srs2effect is ATPase dependent. If a direct physical interactionbetween Srs2 and polymerase delta is necessary, however, ourresults with overexpression of Srs2 in a pol32 ${Delta}$ strain indicatethat Pol32 cannot be the only crucial protein-protein contact(keeping in mind the caveat regarding overexpression).

A second model is that Srs2 and Pol32 inhibit expansions viathe same pathway but without direct contact. Model two is incloser agreement with the Srs2 overexpression study. A thirdpossibility is that expansions arise by a more indirect mechanism,such as defects in checkpoint response associated with lossof Srs2 (23, 46). If true, perhaps Srs2 is involved in a specializedrepair process that responds to replicational stress (23), whichin the case of triplet repeats could be replication fork stalling.Model three does not seem to be mutually exclusive with thefirst two models.

In srs2 rad52 strains, the propensity to expand is similar tothat of an srs2 single mutant. Also, rad51 and rad52 singlemutants show no specific phenotype in our assay. Both findingsare consistent with our interpretation that recombination playsonly a minor role in expansions in our system. In contrast,the srs2 rad51 double mutant showed a larger ( ${approx}$ 3.5-fold) reductionin expansion rates than the srs2 strains, suggesting some specializedeffect of Rad51. Perhaps Rad51 sequesters or inhibits anotherprotein that can partially substitute for Srs2. In rad51 srs2mutants, this other factor might then act to avoid some expansionsthat would otherwise occur due to the absence of Srs2. The identityof this putative second factor is unknown. Alternatively, theabsence of both Srs2 and Rad51 might result in alteration inrepair pathway choice, leading to an effect on expansion rate.This alternate pathway remains hypothetical, however, untilappropriate experiments can be done.

In summary, our results show that the Srs2 helicase acts throughan important new mechanism to provide a selective and potentblock to TNR expansions. These studies provide a paradigm forhow one helicase, Srs2, can suppress expansions.

ACKNOWLEDGMENTS

This work was supported by NIH grant GM61961, by a postdoctoralfellowship from the Huntington's Disease Society of America,and by NIH training grant T32 CA09746.

We thank Hannah Klein for providing strains and plasmids andfor valuable comments on the manuscript, Patrick Sung for providingpurified Srs2 protein, antibodies, and advice on helicase substratedesign, Steve Matson for purified UvrD protein, Luis Marky forguidance on stabilizing the oligonucleotide partial duplex,Alan Morgan for providing an Sgs1-overexpressing plasmid, SergeiMirkin for a pol32 ${Delta}$ strain, and Peter Burgers for anti-Rfc3 antiserum.

FOOTNOTES

* Corresponding author. Mailing address: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Box 986805, Omaha, NE 68198-6805. Phone: (402) 559-4619. Fax: (402) 559-8270. E-mail: rlahue{at}unmc.edu.

REFERENCES

Top