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

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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DNA synthesis errors associated with double-strand-break repair.

Genetics ◽

10.1093/genetics/140.3.965 ◽

1995 ◽

Vol 140 (3) ◽

pp. 965-972 ◽

Cited By ~ 3

Author(s):

J N Strathern ◽

B K Shafer ◽

C B McGill

Keyword(s):

Dna Synthesis ◽

Dna Cleavage ◽

Genome Duplication ◽

Inducible Promoter ◽

Strand Break ◽

Site Specific ◽

Large Populations ◽

Dna Break ◽

A Site ◽

Ho Gene

Abstract Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.

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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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Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4189 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4189-4198 ◽

Cited By ~ 180

Author(s):

G T Milne ◽

S Jin ◽

K B Shannon ◽

D T Weaver

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Strand Break ◽

Yeast Cells ◽

Recombinational Repair ◽

End Joining ◽

Dsb Repair ◽

Dna Double Strand Break ◽

Homologous Recombinational Repair

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.

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Fidelity of Mitotic Double-Strand-Break Repair in Saccharomyces cerevisiae: A Role for SAE2/COM1

Genetics ◽

10.1093/genetics/158.1.109 ◽

2001 ◽

Vol 158 (1) ◽

pp. 109-122 ◽

Cited By ~ 4

Author(s):

Alison J Rattray ◽

Carolyn B McGill ◽

Brenda K Shafer ◽

Jeffrey N Strathern

Keyword(s):

Dna Synthesis ◽

Chromosome Rearrangement ◽

Point Mutations ◽

Nuclease Activity ◽

Repair Process ◽

Strand Break ◽

Recombinational Repair ◽

Physical Analysis ◽

Dna Double Strand Breaks ◽

Dsb Repair

Abstract Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is ∼100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an ∼10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Δ strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.

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Genetic evidence that the meiotic recombination hotspot at the HIS4 locus of Saccharomyces cerevisiae does not represent a site for a symmetrically processed double-strand break.

Genetics ◽

10.1093/genetics/134.1.5 ◽

1993 ◽

Vol 134 (1) ◽

pp. 5-19 ◽

Cited By ~ 5

Author(s):

S E Porter ◽

M A White ◽

T D Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Meiotic Recombination ◽

Strand Break ◽

Yeast Saccharomyces Cerevisiae ◽

Recombination Hotspot ◽

Dna Break ◽

A Site ◽

Aberrant Segregation ◽

His4 Gene

Abstract In the yeast Saccharomyces cerevisiae, the binding of the Rap1 protein to a site located between the 5' end of the HIS4 gene and the 3' end of BIK1 stimulates meiotic recombination at both flanking loci. By using strains that contain mutations located in HIS4 and BIK1, we found that most recombination events stimulated by the binding of Rap1 involve HIS4 or BIK1, rather than bidirectional events including both loci. The patterns of aberrant segregation indicate that most of the Rap1-stimulated recombination events do not represent the symmetric processing of a double-strand DNA break located at the Rap1-binding site.

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

Genetics ◽

10.1093/genetics/143.4.1579 ◽

1996 ◽

Vol 143 (4) ◽

pp. 1579-1587 ◽

Cited By ~ 6

Author(s):

Hiep T Tran ◽

Dmitry A Gordenin ◽

Michael A Resnick

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Mismatch Repair ◽

Dna Polymerase ◽

Recombinational Repair ◽

Null Mutant ◽

Yeast Saccharomyces Cerevisiae ◽

Dna Polymerase Δ ◽

Polymerase Slippage

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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Efficient Incorporation of Large (>2 kb) Heterologies Into Heteroduplex DNA: Pms1/Msh2-Dependent and -Independent Large Loop Mismatch Repair in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.4.1481 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1481-1491 ◽

Cited By ~ 3

Author(s):

Jennifer A Clikeman ◽

Sarah L Wheeler ◽

Jac A Nickoloff

Keyword(s):

Saccharomyces Cerevisiae ◽

Mismatch Repair ◽

Meiotic Recombination ◽

Strand Break ◽

Dsb Repair ◽

Replication Slippage ◽

Heteroduplex Dna ◽

Single Base ◽

Large Loop ◽

Dna Double Strand Break

Abstract DNA double-strand break (DSB) repair in yeast is effected primarily by gene conversion. Conversion can conceivably result from gap repair or from mismatch repair of heteroduplex DNA (hDNA) in recombination intermediates. Mismatch repair is normally very efficient, but unrepaired mismatches segregate in the next cell division, producing sectored colonies. Conversion of small heterologies (single-base differences or insertions <15 bp) in meiosis and mitosis involves mismatch repair of hDNA. The repair of larger loop mismatches in plasmid substrates or arising by replication slippage is inefficient and/or independent of Pms1p/Msh2p-dependent mismatch repair. However, large insertions convert readily (without sectoring) during meiotic recombination, raising the question of whether large insertions convert by repair of large loop mismatches or by gap repair. We show that insertions of 2.2 and 2.6 kbp convert efficiently during DSB-induced mitotic recombination, primarily by Msh2p- and Pms1p-dependent repair of large loop mismatches. These results support models in which Rad51p readily incorporates large heterologies into hDNA. We also show that large heterologies convert more frequently than small heterologies located the same distance from an initiating DSB and propose that this reflects Msh2-independent large loop-specific mismatch repair biased toward loop loss.

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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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Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.6891-6899.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 6891-6899 ◽

Cited By ~ 92

Author(s):

Xuan Wang ◽

Grzegorz Ira ◽

José Antonio Tercero ◽

Allyson M. Holmes ◽

John F. X. Diffley ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

S Phase ◽

Temperature Sensitive ◽

Replication Proteins ◽

Dna Ligases ◽

Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

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