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

Genetics ◽  
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.

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

Genetics ◽  
1996 ◽  
Vol 143 (4) ◽  
pp. 1579-1587 ◽  
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.

scholarly journals Base Composition of Mononucleotide Runs Affects DNA Polymerase Slippage and Removal of Frameshift Intermediates by Mismatch Repair in Saccharomyces cerevisiae

2002 ◽  
Vol 22 (24) ◽  
pp. 8756-8762 ◽  
Author(s):  
Hana Gragg ◽  
Brian D. Harfe ◽  
Sue Jinks-Robertson
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Genome Stability ◽  
The Status ◽  
Nucleotide Insertion ◽  
Mismatch Recognition ◽  
Mutation Assay ◽  
Simple Repeats ◽  
Forward Mutation ◽  
Polymerase Slippage

ABSTRACT The postreplicative mismatch repair (MMR) system is important for removing mutational intermediates that are generated during DNA replication, especially those that arise as a result of DNA polymerase slippage in simple repeats. Here, we use a forward mutation assay to systematically examine the accumulation of frameshift mutations within mononucleotide runs of variable composition in wild-type and MMR-defective yeast strains. These studies demonstrate that (i) DNA polymerase slippage occurs more often in 10-cytosine/10-guanine (10C/10G) runs than in 10-adenine/10-thymine (10A/10T) runs, (ii) the MMR system removes frameshift intermediates in 10A/10T runs more efficiently than in 10C/10G runs, (iii) the MMR system removes −1 frameshift intermediates more efficiently than +1 intermediates in all 10-nucleotide runs, and (iv) the repair specificities of the Msh2p-Msh3p and Msh2p-Msh6p mismatch recognition complexes with respect to 1-nucleotide insertion/deletion loops vary dramatically as a function of run composition. These observations are relevant to issues of genome stability, with both the rates and types of mutations that accumulate in mononucleotide runs being influenced by the primary sequence of the run as well as by the status of the MMR system.

Spontaneous Frameshift Mutations in Saccharomyces cerevisiae: Accumulation During DNA Replication and Removal by Proofreading and Mismatch Repair Activities

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Christopher N Greene ◽  
Sue Jinks-Robertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Wild Type Strain ◽  
Dna Polymerases ◽  
Wild Type ◽  
Triple Mutant ◽  
Repair Capacity ◽  
Frameshift Mutations ◽  
Dna Polymerization ◽  
In The Wild

Abstract The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery. To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2ΔBgl +1 frameshift allele. Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases δ and ε (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed. Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity. Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair. Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast.

scholarly journals Identification of a Mutant DNA Polymerase δ in Saccharomyces cerevisiae With an Antimutator Phenotype for Frameshift Mutations

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 177-186 ◽  
Author(s):  
Michalis I Hadjimarcou ◽  
Robert J Kokoska ◽  
Thomas D Petes ◽  
Linda J Reha-Krantz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Base Substitution ◽  
Msh2 Gene ◽  
Frameshift Mutations ◽  
Frameshift Mutagenesis

Abstract We propose that a β-turn-β structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol δ. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative β-turn-β structure. The mutant DNA pol δ has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol δ was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol δ in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

scholarly journals Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.

1997 ◽  
Vol 17 (5) ◽  
pp. 2859-2865 ◽  
Author(s):  
H T Tran ◽  
J D Keen ◽  
M Kricker ◽  
M A Resnick ◽  
D A Gordenin
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Repair System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genetic Changes ◽  
Coding Sequences ◽  
Frameshift Mutations ◽  
Dna Polymerase Epsilon ◽  
Mismatch Repair System ◽  
Repair Defect

Homonucleotide runs in coding sequences are hot spots for frameshift mutations and potential sources of genetic changes leading to cancer in humans having a mismatch repair defect. We examined frameshift mutations in homonucleotide runs of deoxyadenosines ranging from 4 to 14 bases at the same position in the LYS2 gene of the yeast Saccharomyces cerevisiae. In the msh2 mismatch repair mutant, runs of 9 to 14 deoxyadenosines are 1,700-fold to 51,000-fold, respectively, more mutable for single-nucleotide deletions than are runs of 4 deoxyadenosines. These frameshift mutations can account for up to 99% of all forward mutations inactivating the 4-kb LYS2 gene. Based on results with single and double mutations of the POL2 and MSH2 genes, both DNA polymerase epsilon proofreading and mismatch repair are efficient for short runs while only the mismatch repair system prevents frameshift mutations in runs of > or = 8 nucleotides. Therefore, coding sequences containing long homonucleotide runs are likely to be at risk for mutational inactivation in cells lacking mismatch repair capability.

scholarly journals The 3′→5′ Exonucleases of DNA Polymerases δ and ɛ and the 5′→3′ Exonuclease Exo1 Have Major Roles in Postreplication Mutation Avoidance in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (3) ◽  
pp. 2000-2007 ◽  
Author(s):  
Hiep T. Tran ◽  
Dmitry A. Gordenin ◽  
Michael A. Resnick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Mutation Rate ◽  
Mutation Detection ◽  
Dna Polymerases ◽  
Genetic Interactions ◽  
Detection Systems ◽  
Mutation Avoidance ◽  
Highly Sensitive

ABSTRACT Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5′→3′ Exo1 and the 3′→5′ DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase ɛ (pol2-4) and δ (pol3-01) mutants defective in 3′→5′ proofreading exonuclease, mutants defective in the 5′→3′ exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.

scholarly journals Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

2017 ◽  
Vol 114 (14) ◽  
pp. 3607-3612 ◽  
Author(s):  
Nikki Bowen ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Strand Break ◽  
Single Strand Break ◽  
Mutl Homolog 1 ◽  
Exonuclease 1 ◽  
Postmeiotic Segregation ◽  
Mmr Proteins

Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)–MutS homolog 6 (Msh6) or the Msh2–MutS homolog 3 (Msh3) stimulates 5′ to 3′ excision by exonuclease 1 (Exo1) from a single-strand break 5′ to the mispair, excising the mispair. In the other, Msh2–Msh6 or Msh2–Msh3 activate the MutL homolog 1 (Mlh1)–postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3′ to the mispair, to make nicks 5′ to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase ε (Pol ε) may also play a role in MMR. Here we describe the reconstitution of Pol ε-dependent MMR using S. cerevisiae proteins. A mixture of Msh2–Msh6 (or Msh2–Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze both short-patch and long-patch 5′ nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3′ to the mispair, a mixture of Msh2–Msh6 (or Msh2–Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze an MMR reaction that required Mlh1–Pms1. These results demonstrate that Pol ε can act in eukaryotic MMR in vitro.

A Mutation in a Saccharomyces cerevisiae Gene (RAD3) Required for Nucleotide Excision Repair and Transcription Increases the Efficiency of Mismatch Correction

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 459-466 ◽  
Author(s):  
Yingying Yang ◽  
Anthony L Johnson ◽  
Leland H Johnston ◽  
Wolfram Siede ◽  
Errol C Friedberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Surprising Result ◽  
Spontaneous Mutation Rate ◽  
Mismatch Correction ◽  
Nucleotide Excision ◽  
Repair Genes ◽  
Type Strains

Abstract RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMSl, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase δ was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.

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