Meiotic recombination between dispersed repeated genes is associated with heteroduplex formation.

In Saccharomyces cerevisiae, recombination events occurring between allelic genes located on homologous chromosomes are often associated with heteroduplex formation. We found that recombination events between repeated genes on nonhomologous chromosomes (ectopic events) are also associated with the formation of heteroduplexes, indicating that classical and ectopic recombination events involve similar mechanisms

Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae.

Heteroduplexes formed between DNA strands derived from different homologous chromosomes are an intermediate in meiotic crossing over in the yeast Saccharomyces cerevisiae and other eucaryotes. A heteroduplex formed between wild-type and mutant genes will contain a base pair mismatch; failure to repair this mismatch will lead to postmeiotic segregation (PMS). By analyzing the frequency of PMS for various mutant alleles in the yeast HIS4 gene, we showed that C/C mismatches were inefficiently repaired relative to all other point mismatches. These other mismatches (G/G, G/A, T/T, A/A, T/C, C/A, A/A, and T/G) were repaired with approximately the same efficiency. We found that in spores with unrepaired mismatches in heteroduplexes, the nontranscribed strand of the HIS4 gene was more frequently donated than the transcribed strand. In addition, the direction of repair for certain mismatches was nonrandom

Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae.

The full activity of a recombination initiation site located 5' of HIS4 requires the binding of the transcription factors RAP1, BAS1, and BAS2. Two RAP1 binding sites can substitute for the wild-type initiation site. A 51-bp region of telomeric DNA inserted upstream of either HIS4 or ARG4 very strongly stimulates recombination. We suggest that the ability of transcription factors to induce recombination is a consequence of an altered chromatin structure that favors the entry of proteins that initiate recombination, rather than an effect of these factors on transcription

Low Levels of DNA Polymerase Alpha Induce Mitotic and Meiotic Instability in the Ribosomal DNA Gene Cluster of Saccharomyces cerevisiae

The ribosomal DNA (rDNA) genes of Saccharomyces cerevisiae are located in a tandem array of about 150 repeats. Using a diploid with markers flanking and within the rDNA array, we showed that low levels of DNA polymerase alpha elevate recombination between both homologues and sister chromatids, about five-fold in mitotic cells and 30-fold in meiotic cells. This stimulation is independent of Fob1p, a protein required for the programmed replication fork block (RFB) in the rDNA. We observed that the fob1 mutation alone significantly increased meiotic, but not mitotic, rDNA recombination, suggesting a meiosis-specific role for this protein. We found that meiotic cells with low polymerase alpha had decreased Sir2p binding and increased Spo11p-catalyzed double-strand DNA breaks in the rDNA. Furthermore, meiotic crossover interference in the rDNA is absent. These results suggest that the hyper-Rec phenotypes resulting from low levels of DNA polymerase alpha in mitosis and meiosis reflect two fundamentally different mechanisms: the increased mitotic recombination is likely due to increased double-strand DNA breaks (DSBs) resulting from Fob1p-independent stalled replication forks, whereas the hyper-Rec meiotic phenotype results from increased levels of Spo11-catalyzed DSBs in the rDNA

Increased Rates of Genomic Deletions Generated by Mutations in the Yeast Gene Encoding DNA Polymerase delta or by Decreases in the Cellular Levels of DNA Polymerase delta

In Saccharomyces cerevisiae, POL3 encodes the catalytic subunit of DNA polymerase δ. While yeast POL3 mutant strains that lack the proofreading exonuclease activity of the polymerase have a strong mutator phenotype, little is known regarding the role of other Pol3p domains in mutation avoidance. We identified a number of pol3 mutations in regions outside of the exonuclease domain that have a mutator phenotype, substantially elevating the frequency of deletions. These deletions appear to reflect an increased frequency of DNA polymerase slippage. In addition, we demonstrate that reduction in the level of wild-type DNA polymerase results in a similar mutator phenotype. Lowered levels of DNA polymerase also result in increased sensitivity to the DNA-damaging agent methyl methane sulfonate. We conclude that both the quantity and the quality of DNA polymerase δ is important in ensuring genome stability

Ninety-Six Haploid Yeast Strains With Individual Disruptions of Open Reading Frames Between YOR097C and YOR192C, Constructed for the Saccharomyces Genome Deletion Project, Have an Additional Mutation in the Mismatch Repair Gene MSH3

As part of the Saccharomyces Genome Deletion Project, sets of presumably isogenic haploid and diploid strains that differed only by single gene deletions were constructed. We found that one set of 96 strains (containing deletions of ORFs located between YOR097C and YOR192C) in the collection, which was derived from the haploid BY4741, has an additional mutation in the MSH3 mismatch repair gene

Triplet repeats form secondary structures that escape DNA repair in yeast

Several human neurodegenerative diseases result from expansion of CTG/CAG or CGG/CCG triplet repeats. The finding that single-stranded CNG repeats form hairpin-like structures in vitro has led to the hypothesis that DNA secondary structure formation is an important component of the expansion mechanism. We show that single-stranded DNA loops containing 10 CTG/CAG or CGG/CCG repeats are inefficiently repaired during meiotic recombination in Saccharomyces cerevisiae. Comparisons of the repair of DNA loops with palindromic and nonpalindromic sequences suggest that this inefficient repair reflects the ability of these sequences to form hairpin structures in vivo

Nonrandom Distribution of Interhomolog Recombination Events Induced by Breakage of a Dicentric Chromosome in Saccharomyces cerevisiae

Dicentric chromosomes undergo breakage in mitosis, resulting in chromosome deletions, duplications, and translocations. In this study, we map chromosome break sites of dicentrics in Saccharomyces cerevisiae by a mitotic recombination assay. The assay uses a diploid strain in which one homolog has a conditional centromere in addition to a wild-type centromere, and the other homolog has only the wild-type centromere; the conditional centromere is inactive when cells are grown in galactose and is activated when the cells are switched to glucose. In addition, the two homologs are distinguishable by multiple single-nucleotide polymorphisms (SNPs). Under conditions in which the conditional centromere is activated, the functionally dicentric chromosome undergoes double-stranded DNA breaks (DSBs) that can be repaired by mitotic recombination with the homolog. Such recombination events often lead to loss of heterozygosity (LOH) of SNPs that are centromere distal to the crossover. Using a PCR-based assay, we determined the position of LOH in multiple independent recombination events to a resolution of ∼4 kb. This analysis shows that dicentric chromosomes have recombination breakpoints that are broadly distributed between the two centromeres, although there is a clustering of breakpoints within 10 kb of the conditional centromere