High-resolution mapping of homologous recombination events in rad3 hyper-recombination mutants. Saccharomyces cerevisiae

The Saccharomyces cerevisae RAD3 gene is homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Mutant alleles of RAD3 have been identified (rad3-101 and rad3-102) that have partial defects in DNA repair associated with 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. We have further characterized these events using a system in which the reciprocal products of mitotic recombination between homologs are recovered as red and white sectored colonies. Both rad3-101 and rad3-102 elevate the frequency of sectored colonies about 100-fold. Subsequent mapping of these events shows that three-quarters of crossovers between homologs induced in hyper-Rec rad3 mutants reflect DSBs formed in 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. In addition to examining crossovers on chromosome V, 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. Overall design: This experiment involves the analysis of 96 two-color microarrays. 86 of the microarrays represent the analysis of 43 red/white sectored colonies in which the sectors have the reciprocal products of a mitotic recombination event on chromosome V. In 68 of these 86 microarrays, we used SNP arrays in which recombination events throughout the genome could be analyzed (whole-genome arrays; abbreviated WG in the files below). The strains used in this analysis (MD555 and MD556) were independent isogenic strains that were homozygous for the rad3-102 mutation; 40 arrays were examined for MD555 and 26 arrays were examined for MD556. We also examined ten sectored colonies derived from the strain SLA64 (20 of 86 microarrays). This strain is identical to MD555 and MD556 except that it is homozygous for the rad3-101 allele instead of the rad3-102 allele .For two of these sectored colonies (four arrays), we used the whole genome microarrays. For the other eight sectored colonies (16 microarrays), we used microarrays that had dense arrays of oligonucleotides specific for chromosomes I, III, V, and the right arm of chromosome VIII; in addition, the arrays had oligonucleotides representing the telomeres and centromeres of the other yeast chromosomes. Below, we use the designation VA (chromosome V-specific arrays) in the file names. Finally, we examined ten whole-genome arrays in which sub-cultured strains were analyzed; five were derived from MD555, and five from SLA64. For all experiments, genomic DNA from the experimental strain was labeled with Cy5-dUTP and from the control isogenic wild-type diploids (PG311 or JSC23, as indicated below) with Cy3-dUTP. The strains PG311 and JSC23 are described in detail in Lee et al. (2009) PLoS Genetics 5: e1000410 and Yin and Petes (2013) PLoS Genetics 9: 1003894, respectively.