Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers

High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast.

In yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist. Most previous studies involving hetDNA formed during mitotic recombination were restricted to one locus. Below, we present a global mapping of hetDNA formed in the MMR-defective mlh1 strain. We find that many recombination events are associated with repair of double-stranded DNA gaps and/or involve Mlh1-independent mismatch repair. Many of our events are not explicable by the simplest form of the double-strand break repair model of recombination

The stabilization of repetitive tracts of DNA by variant repeats requires a functional DNA mismatch repair system

AbstractSimple repetitive tracts of DNA are unstable in all organisms thus far examined. In the yeast S. cerevisiae, we show that a 51 by poly(GT) tract alters length at a rate of about 10−5 per cell division. Insertion of a single variant repeat (either AT or CT) into the middle of the poly(GT) tract results in 100-fold stabilization. This stabilization requires the DNA mismatch repair system. Alterations within tracts with variant repeats occur more frequently on one side of the interruption than on the other. The stabilizing effects of variant repeats and polarity of repeat alterations have also been observed in trinucleotide repeats associated with certain human diseases

Context dependence of meiotic recombination hotspots in yeast: the relationship between recombination activity of a reporter construct and base composition.

Borde and colleagues reported that a reporter plasmid inserted at different genomic locations in Saccharomyces cerevisiae had different levels of meiotic recombination activity. We show that the level of recombination activity is very significantly correlated with the GC content of DNA sequences flanking the insertion

Haploidization in Saccharomyces cerevisiae Induced by a Deficiency in Homologous Recombination

Diploid Saccharomyes cerevisae strains lacking the RAD52 gene required for homologous recombination have a very high rate of chromosome loss. Two of four isolates subcultured ∼20 times (∼500 cell divisions) became haploid. These strains were capable of mating with wild-type haploids to produce diploid progeny capable of undergoing meiosis to produce four viable spores

Dependence of the regulation of telomere length on the type of subtelomeric repeat in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, chromosomes terminate with approximately 400 bp of a simple repeat poly(TG(1-3)). Based on the arrangement of subtelomeric X and Y' repeats, two types of yeast telomeres exist, those with both X and Y' (Y' telomeres) and those with only X (X telomeres). Mutations that result in abnormally short or abnormally long poly(TG(1-3)) tracts have been previously identified. In this study, we investigated telomere length in strains with two classes of mutations, one that resulted in short poly(TG(1-3)) tracts (tel1) and one that resulted in elongated tracts (pif1, rap1-17, rif1, or rif2). In the tel1 pif1 strain, Y' telomeres had about the same length as those in tel1 strains and X telomeres had lengths intermediate between those in tel1 and pif1 strains. Strains with either the tel1 rap1-17 or tel1 rif2 genotypes had short tracts for all chromosome ends examined, demonstrating that the telomere elongation characteristic of rap1-17 and rif2 strains is Tel1p-dependent. In strains of the tel1 rif1 or tel1 rif1 rif2 genotypes, telomeres with Y' repeats had short terminal tracts, whereas most of the X telomeres had long terminal tracts. These results demonstrate that the regulation of telomere length is different for X and Y' telomeres

Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae.

DNA fragments (generated by BamHI treatment) with no homology to the yeast genome were transformed into Saccharomyces cerevisiae. When the fragments were transformed in the presence of the BamHI enzyme, they integrated into genomic BamHI sites. When the fragments were transformed in the absence of the enzyme, they integrated into genomic G-A-T-C sites. Since the G-A-T-C sequence is present at the ends of BamHI fragments, this result indicates that four base pairs of homology are sufficient for some types of mitotic recombinatio

Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae

One important source of genomic instability associated with tumor cells is DNA replication stress. In the current study, replication stress was induced in yeast by a 10-fold reduction in the level of the replicative DNA polymerase δ. By DNA microarray analysis and high-throughput DNA sequencing, we showed that this stress resulted in very high rates of both large (aneuploidy, mitotic recombination, deletions and duplications, and translocations) and small (point mutations and small insertion/deletions) genetic alterations. Some of these changes resulted in a selective growth advantage of the cells, demonstrating the role of elevated genetic instability in the rapid evolution of cells in challenging growth conditions

High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast

The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.España, Ministerio de Economía y Competitividad BFU2010-16372España, Ministerio de Economía y Competitividad BFU2013-42918-