Accumulation of Recessive Lethal Mutations in <i>Saccharomyces cerevisiae mlh1</i> Mismatch Repair Mutants Is Not Associated With Gross Chromosomal Rearrangements

Heck, Julie A.; Gresham, David; Botstein, David; Alani, Eric

doi:10.1534/genetics.106.059311

Cited by 11 publications

(20 citation statements)

References 24 publications

(25 reference statements)

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“…Such a phenotype allowed us to easily identify recessive lethal mutations (Heck et al 2006). At the nonpermissive temperature, the mlh1-7 ts mutation conferred a phenotype similar to the null in the canavanine resistance mutation assay and a mutator phenotype in the lys2-A14 reversion assay that was 1000-fold higher than MLH1 but 4-fold lower than the null (Heck et al 2006); J. Heck and E. Alani, unpublished observations).…”

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confidence: 99%

“…The baker's yeast S. cerevisiae is an ideal model system in which to perform these studies because genetic analysis of many of the key MMR factors has been performed; more importantly the effect of null mutations in these factors has been extensively characterized using a variety of mutator assays (Kunkel and Erie 2005). Previously, one of our groups (Heck et al 2006) grew wild-type and conditional mlh1 (mlh1-7 ts ) diploid strains of S. cerevisiae for 160 generations with bottlenecks that reduced the population size to one cell every 20 generations. These lines were grown at 35°, the nonpermissive temperature for mlh1-7 ts .…”

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“…In contrast, mlh1-7 ts lines displayed spore viabilities that ranged from 1.1 to 77%, demonstrating that the lines had accumulated recessive lethal mutations. Second, comparative genome hybridization (CGH) and pulse-field (PFGE) analyses of the mlh1-7 ts strains indicated that they did not undergo major genome rearrangements (Heck et al 2006). Third, because the lines were grown as diploids for a limited number of generations, secondary mutations, dominant or recessive, that alter the rate or type of mutagenesis should rarely occur.…”

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confidence: 99%

“…Also, because there is no sexual reproduction and mutations should clonally propagate after escaping the initial bottleneck, newly arising mutations should appear as heterozygous sites. Finally, the above strategy should limit biases in mutation accumulation because the diploid cells were grown in rich media under minimal selection pressure where deleterious mutations could accumulate (Heck et al 2006).…”

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confidence: 99%

“…Together this work provides new insights into how mismatch repair can shape genome stability and dynamics, mutation mechanisms, and evolution. (Heck et al 2006). Three of the 10 mlh1-7 ts lines at generation 160 were analyzed by wholegenome sequencing.…”

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confidence: 99%

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Detection of Heterozygous Mutations in the Genome of Mismatch Repair Defective Diploid Yeast Using a Bayesian Approach

Zanders

RoyChoudhury

et al. 2010

Genetics

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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. M UTATION rates in prokaryotic and eukaryotic organisms are typically determined by measuring reversion or forward mutation for specific marker alleles. These values are then extrapolated to obtain genomewide estimates. Mutation rates in higher eukaryotes are also estimated by analyzing sequence divergence between different strains or species, followed by reconstructing the accumulation of mutations since divergence (reviewed in Nishant et al. 2009). These approaches suffer from two main limitations. First, recent studies have shown that mutation rate and repair efficiency vary across the genome and are affected by parameters that include base composition, local recombination rate, gene density, transcriptional activity, repair efficiency, chromatin structure, nucleosome position, and replication timing (Wolfe et al. 1989; Datta and JinksRobertson 1995;Matassi et al. 1999;Hardison et al. 2003;Arndt et al. 2005;Hawk et al. 2005;Teytelman et al. 2008;Washietl ...

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Detection of Heterozygous Mutations in the Genome of Mismatch Repair Defective Diploid Yeast Using a Bayesian Approach

Zanders

RoyChoudhury

et al. 2010

Genetics

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Genomic mutation rates: what high‐throughput methods can tell us

2009

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Summary High-throughput DNA analyses are increasingly being used to detect rare mutations in moderately sized genomes. These methods have yielded genome mutation rates that are markedly higher than those obtained using pre-genomic strategies. Recent work in a variety of organisms has shown that mutation rate is strongly affected by sequence context and genome position. These observations suggest that high-throughput DNA analyses will ultimately allow researchers to identify trans-acting factors and cis sequences that underlie mutation rate variation. Such work should provide insights on how mutation rate variability can impact genome organization and disease progression.

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Accumulation of Recessive Lethal Mutations in Saccharomyces cerevisiae mlh1 Mismatch Repair Mutants Is Not Associated With Gross Chromosomal Rearrangements

Cited by 11 publications

References 24 publications

Detection of Heterozygous Mutations in the Genome of Mismatch Repair Defective Diploid Yeast Using a Bayesian Approach

Detection of Heterozygous Mutations in the Genome of Mismatch Repair Defective Diploid Yeast Using a Bayesian Approach

Genomic mutation rates: what high‐throughput methods can tell us

Current awareness on yeast

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