A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae (original) (raw)
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G3 (Bethesda, Md.), 2013
DNA mismatch repair is a highly conserved DNA repair pathway. In humans, germline mutations in hMSH2 or hMLH1, key components of mismatch repair, have been associated with Lynch syndrome, a leading cause of inherited cancer mortality. Current estimates of the mutation rate and the mutational spectra in mismatch repair defective cells are primarily limited to a small number of individual reporter loci. Here we use the yeast Saccharomyces cerevisiae to generate a genome-wide view of the rates, spectra, and distribution of mutation in the absence of mismatch repair. We performed mutation accumulation assays and next generation sequencing on 19 strains, including 16 msh2 missense variants implicated in Lynch cancer syndrome. The mutation rate for DNA mismatch repair null strains was approximately 1 mutation per genome per generation, 225-fold greater than the wild-type rate. The mutations were distributed randomly throughout the genome, independent of replication timing. The mutation sp...
DNA replication errors that escape polymerase proofreading and mismatch repair (MMR) can lead to base substitution and frameshift mutations. Such mutations can disrupt gene function, reduce fitness, and promote diseases such as cancer and are also the raw material of molecular evolution. To analyze with limited bias genomic features associated with DNA polymerase errors, we performed a genome-wide analysis of mutations that accumulate in MMR-deficient diploid lines of Saccharomyces cerevisiae. These lines were derived from a common ancestor and were grown for 160 generations, with bottlenecks reducing the population to one cell every 20 generations. We sequenced to between 8-and 20-fold coverage one wildtype and three mutator lines using Illumina Solexa 36-bp reads. Using an experimentally aware Bayesian genotype caller developed to pool experimental data across sequencing runs for all strains, we detected 28 heterozygous single-nucleotide polymorphisms (SNPs) and 48 single-nt insertion/deletions (indels) from the data set. This method was evaluated on simulated data sets and found to have a very low false-positive rate ($6 3 10 À5 ) and a false-negative rate of 0.08 within the unique mapping regions of the genome that contained at least sevenfold coverage. The heterozygous mutations identified by the Bayesian genotype caller were confirmed by Sanger sequencing. All of the mutations were unique to a given line, except for a single-nt deletion mutation which occurred independently in two lines. All 48 indels, composed of 46 deletions and two insertions, occurred in homopolymer (HP) tracts [i.e., 47 poly(A) or (T) tracts, 1 poly(G) or (C) tract] between 5 and 13 bp long. Our findings are of interest because HP tracts are present at high levels in the yeast genome (.77,400 for 5-to 20-nt HP tracts), and frameshift mutations in these regions are likely to disrupt gene function. In addition, they demonstrate that the mutation pattern seen previously in mismatch repair defective strains using a limited number of reporters holds true for the entire genome.
A genome-wide view of the spectrum of spontaneous mutations in yeast
Proceedings of the National Academy of Sciences, 2008
The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.
From the Cover: A genome-wide view of the spectrum of spontaneous mutations in yeast
Proceedings of The National Academy of Sciences, 2008
The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.
The Origin of Spontaneous Mutation in Saccharomyces Cerevisiae
Genetics, 1980
Characterization of two antimutator loci in yeast shows that both are members of the same mutagenic repair system known to be responsible for almost all induced mutation (Lawrence and Christensen 1976, 1979a,b; Prakash 1976). One of the these newly isolated antimutator mutations is an allele of rev3 (Lemontt 1971b). Two other alleles of rev3 were tested and were also found to be antimutators. Double mutants carrying rev3 and mutator mutations of rad3, rad51 or rad18 are like rev3 single mutants with respect to spontaneous mutation rate, supporting the hypothesis (Hastings, Quah and von Borstel 1976) that many mutators in yeast act by channelling spontaneous lesions from accurate to mutagenic repair. However, the enhanced mutation rate seen in a radiation-resistant mutator mutant mut1 is not dependent on REV3, but is dependent on another gene designated ANT1. An additive effect on the reduction in spontaneous mutation, seen in the ant1 rev3 double-mutant strain, leads to the conclusi...
Yeast, 1999
The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7, . When compared to wild-type, ogg1 mutants show an increase in the frequency of GC to TA transversions, indicating a role for Ogg1 in the repair of 8-OxoG. Here we report an increased frequency of forward mutation to canavanine resistance in mutants defective in the nucleotide excision repair (NER) gene RAD14. This was not increased further in strains additionally defective in OGG1. However, when compared to strains solely defective in OGG1, ogg1rad14 mutants displayed an increase in spontaneous GC to TA transversions. Intriguingly, reversion of the lys1-1 ochre allele was not increased in rad14 mutants, suggesting that oxidative base damage may only represent a substrate for NER in certain regions of the genome. We also examined repair of oxidative DNA damage by transforming mutant strains with plasmid DNA treated with methylene blue plus visible light. Mutants defective in OGG1 showed no significant reduction in transformation efficiency compared with wild-type strains. In contrast, disruption of RAD14 reduced the efficiency of transformation, yet there was no further decrease in an ogg1rad14 mutant. This strongly supports a role for NER in the repair of oxidative base damage in yeast, and differs from similar experiments carried out in E. coli, where transformation efficiency is only reduced in mutants defective in both fpg and uvrA. Finally, the repair of Fpg-sensitive sites was examined at the MAT and HML mating type loci, and NER was found to play a role in their removal.
Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae
Biochemical and Biophysical Research Communications, 2004
To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 • 10 À7 , 100-fold lower than that in diploid cells (1.03 • 10 À4). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C fi A:T and G:C fi T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.
Proceedings of the National Academy of Sciences of the United States of America, 2003
A genomewide screen of a collection of 4,847 yeast gene deletion mutants was carried out to identify the genes required for suppressing mutations in the CAN1 forward-mutation assay. The primary screens and subsequent analysis allowed (i) identification of 18 known mutator mutants, providing a solid means for checking the efficiency of the screen, and (ii) identification of a number of genes not known previously to be involved in suppressing mutations. Among the previously uncharacterized mutation-suppressing genes were six genes of unknown function including four (CSM2, SHU2, SHU1, and YLR376c) encoding proteins that interact with each other and promote resistance to killing by methyl methanesulfonate, one gene (EGL1) previously identified as suppressing Ty1 mobility and recombination between repeated sequences, and one gene (YLR154c) that was not associated with any known processes. In addition, five genes (TSA1, SOD1, LYS7, SKN7, and YAP1) implicated in the oxidative-stress responses were found to play a significant role in mutation suppression. Furthermore, TSA1, which encodes thioredoxin peroxidase, was found to strongly suppress gross chromosomal rearrangements. These results provide a global view of the nonessential genes involved in preventing mutagenesis. Study of such genes should provide useful clues in identification of human genes potentially involved in cancer predisposition and in understanding their mechanisms of action.