The Prevention of Repeat-Associated Deletions in Saccharomyces cerevisiae by Mismatch Repair Depends on Size and Origin of Deletions

Abstract We have investigated the effects of mismatch repair on 1- to 61-bp deletions in the yeast Saccharomyces cerevisiae. The deletions are likely to involve unpaired loop intermediates resulting from DNA polymerase slippage. The mutator effects of mutations in the DNA polymerase δ (POL3) gene and the recombinational repair RAD52 gene were studied in combination with mismatch repair defects. The pol3-t mutation increased up to 1000-fold the rate of extended (7-61 bp) but not of 1-bp deletions. In a rad52 null mutant only the 1-bp deletions were increased (12-fold). The mismatch repair mutations pmsl, msh2 and msh3 did not affect 31- and 61-bp deletions in the pol3-t but increased the rates of 7- and 1-bp deletions. We propose that loops less than or equal to seven bases generated during replication are subject to mismatch repair by the PMSI, MSH2, MSH3 system and that it cannot act on loops ≤31 bases. In contrast to the pol3-t, the enhancement of 1-bp deletions in a rad52 mutant is not altered by a pmsl mutation. Thus, mismatch repair appears to be specific to errors of DNA synthesis generated during semiconservative replication.

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Sequence Composition and Context Effects on the Generation and Repair of Frameshift Intermediates in Mononucleotide Runs in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.2.571 ◽

2000 ◽

Vol 156 (2) ◽

pp. 571-578

Author(s):

Brian D Harfe ◽

Sue Jinks-Robertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Mismatch Repair ◽

Dna Polymerase ◽

Context Effects ◽

Complete Elimination ◽

Sequence Context ◽

Frameshift Mutations ◽

Sequence Composition ◽

Overall Efficiency ◽

Polymerase Slippage

Abstract DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations. In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase. Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR. To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles. Slippage events within these runs were detected as Lys+ revertants. 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context. Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.

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The Chromosome Bias of Misincorporations During Double-Strand Break Repair Is Not Altered in Mismatch Repair–Defective Strains of Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/148.4.1525 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1525-1533 ◽

Cited By ~ 3

Author(s):

Carolyn B McGill ◽

Susan L Holbeck ◽

Jeffrey N Strathern

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Mismatch Repair ◽

Strand Break ◽

Recombinational Repair ◽

Dsb Repair ◽

Site Specific ◽

Initial Sequence ◽

Dna Break ◽

A Site

AbstractRecombinational repair of a site-specific, double-strand DNA break (DSB) results in increased reversion frequency for nearby mutations. Although some models for DSB repair predict that newly synthesized DNA will be inherited equally by both the originally broken chromosome and the chromosome that served as a template, the DNA synthesis errors are almost exclusively found on the chromosome that had the original DSB (introduced by the HO endonuclease). To determine whether mismatch repair acts on the template chromosome in a directed fashion to restore mismatches to the initial sequence, these experiments were repeated in mismatch repair-defective (pms1, mlh1, and msh2) backgrounds. The results suggest that mismatch repair is not responsible for the observed bias.

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The Conversion Gradient at HIS4 of Saccharomyces cerevisiae. II. A Role for Mismatch Repair Directed by Biased Resolution of the Recombinational Intermediate

Genetics ◽

10.1093/genetics/153.2.573 ◽

1999 ◽

Vol 153 (2) ◽

pp. 573-583 ◽

Cited By ~ 10

Author(s):

Henriette M Foss ◽

Kenneth J Hillers ◽

Franklin W Stahl

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Mismatch Repair ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Holliday Junction ◽

Crossing Over ◽

Gradient Type ◽

Salient Features

AbstractSalient features of recombination at ARG4 of Saccharomyces provoke a variation of the double-strand-break repair (DSBR) model that has the following features: (1) Holliday junction cutting is biased in favor of strands upon which DNA synthesis occurred during formation of the joint molecule (this bias ensures that cutting both junctions of the joint-molecule intermediate arising during DSBR usually leads to crossing over); (2) cutting only one junction gives noncrossovers; and (3) repair of mismatches that are semirefractory to mismatch repair and/or far from the DSB site is directed primarily by junction resolution. The bias in junction resolution favors restoration of 4:4 segregation when such mismatches and the directing junction are on the same side of the DSB site. Studies at HIS4 confirmed the predicted influence of the bias in junction resolution on the conversion gradient, type of mismatch repair, and frequency of aberrant 5:3 segregation, as well as the predicted relationship between mismatch repair and crossing over.

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A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽

10.1093/genetics/153.2.595 ◽

1999 ◽

Vol 153 (2) ◽

pp. 595-605 ◽

Cited By ~ 2

Author(s):

Bradley J Merrill ◽

Connie Holm

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Synthetic Lethality ◽

Velocity Sedimentation ◽

Recombinational Repair ◽

Repair Pathway ◽

Dna Breaks ◽

Single Stranded Dna ◽

Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

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A Mutation of the Yeast Gene Encoding PCNA Destabilizes Both Microsatellite and Minisatellite DNA Sequences

Genetics ◽

10.1093/genetics/151.2.511 ◽

1999 ◽

Vol 151 (2) ◽

pp. 511-519 ◽

Cited By ~ 4

Author(s):

Robert J Kokoska ◽

Lela Stefanovic ◽

Andrew B Buermeyer ◽

R Michael Liskay ◽

Thomas D Petes

Keyword(s):

Dna Polymerase ◽

Repetitive Dna ◽

Dna Sequences ◽

Repeat Unit ◽

Nuclear Antigen ◽

Temperature Sensitive ◽

Dinucleotide Repeats ◽

Yeast Saccharomyces Cerevisiae ◽

Dna Polymerase Δ ◽

Repeat Units

AbstractThe POL30 gene of the yeast Saccharomyces cerevisiae encodes the proliferating cell nuclear antigen (PCNA), a protein required for processive DNA synthesis by DNA polymerase δ and ϵ. We examined the effects of the pol30-52 mutation on the stability of microsatellite (1- to 8-bp repeat units) and minisatellite (20-bp repeat units) DNA sequences. It had previously been shown that this mutation destabilizes dinucleotide repeats 150-fold and that this effect is primarily due to defects in DNA mismatch repair. From our analysis of the effects of pol30-52 on classes of repetitive DNA with longer repeat unit lengths, we conclude that this mutation may also elevate the rate of DNA polymerase slippage. The effect of pol30-52 on tracts of repetitive DNA with large repeat unit lengths was similar, but not identical, to that observed previously for pol3-t, a temperature-sensitive mutation affecting DNA polymerase δ. Strains with both pol30-52 and pol3-t mutations grew extremely slowly and had minisatellite mutation rates considerably greater than those observed in either single mutant strain.

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