title: High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, United States of America.
authors.forename: Sabrina L
authors.initials: SL
authors.lastname: Andersen
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, United States of America.
authors.forename: Aimee
authors.initials: A
authors.lastname: Zhang
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, United States of America.
authors.forename: Margaret
authors.initials: M
authors.lastname: Dominska
authors.affiliation: Department of Molecular Biology, Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER-Universidad de Sevilla, Seville, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Moriel-Carretero
authors.affiliation: Department of Molecular Biology, Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER-Universidad de Sevilla, Seville, Spain.
authors.forename: Emilia
authors.initials: E
authors.lastname: Herrera-Moyano
authors.affiliation: Department of Molecular Biology, Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER-Universidad de Sevilla, Seville, Spain.
authors.forename: Andrés
authors.initials: A
authors.lastname: Aguilera
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, United States of America.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Petes
mesh_terms: D002842:Chromatids / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics; D059765:Homologous Recombination / Q000235:genetics*; D006801:Humans; D008938:Mitosis / Q000235:genetics; D050505:Mutant Proteins / Q000235:genetics; D010641:Phenotype; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases