A genomic screen in Saccharomyces cerevisiae to identify gene deletions that affect Rad52 focus formation.

摘要

The repair of DNA damage and the maintenance of genomic integrity are essential to the viability of all cells. Different types of DNA damage require unique pathways for efficient and appropriate repair. DNA double-strand breaks and single-strand nicks encountered during replication are repaired primarily through the process of homologous recombination, which utilizes the genetic information in homologous DNA sequences to repair DNA lost at the site of the lesion. Homologous recombination in eukaryotes is mediated by proteins in the Rad52 epistasis group, which has been extensively studied through genetic and biochemical means. Application of cell biology techniques and fluorescent protein fusions has permitted an examination of the dynamics of Rad52 group proteins in living cells, revealing the reorganization of the proteins from diffuse nuclear localizations to discrete subnuclear foci reflecting repair centers actively engaged in homologous recombination, in response to both exogenous DNA damage and endogenous lesions encountered during replication.; To explore the contribution of yeast genes to pathways of DNA integrity and homologous recombination itself, we took advantage of the yeast gene deletion library to score deletions of all non-essential genes for their effects on the spontaneous formation of Rad52-YFP foci. In contrast to traditional screens that assay DNA repair and homologous recombination by measuring the products of recombination events or cell survival following DNA damage, our cell biology approach permitted the inspection of mutants that affect the incidence and dynamics of focus formation regardless of the recombination outcome. An initial screen performed by transforming a plasmid containing the RAD32-YFP fusion gene directly into library haploids yielded an excessive number of false positive results, which were determined to be the result of additional recessive factors in the library strains. To isolate the marked gene deletion in each strain from other recessive alleles, we developed a method, synthetic hybrid loss of heterozygosity, which takes advantage of a set of sixteen outbred strains each of which permits the selectable loss of one of the yeast chromosomes. Mating the library strains to the appropriate conditional chromosome strain reduces the contribution of other genetic factors to the Rad52 focus phenotype through complementation, while forcing the diploid to lose the chromosome homologous to the one bearing the marked gene deletion achieves homozygosity at that locus, allowing the gene deletions to be assayed as hybrid diploids. We demonstrate that this method significantly reduces the levels of false positive and negative results in the Rad52-YFP focus screen.; Upon examination of the complete set of gene deletions, we identified 86 gene deletions that lead to increases in the levels of spontaneous foci in proliferating cells, 22 of which were deletions of previously uncharacterized ORFs. The genes identified in this work, including the newly identified ones, demonstrate considerable conservation throughout eukaryotic evolution thus allowing the prediction of function for these genes in other organisms as well. In this set of mutants, we measured the rates of spontaneous recombination between sister chromatids and between chromosome homologs as well as synthetic genetic interactions between each mutant and rad523. Subsequent analysis allowed us to parse the 86 gene deletions into 4 classes that reflect a diversity of mechanisms that can generate the focus phenotype. In addition to mutations that increase the formation of spontaneous lesions or that block the completion of recombination, we found that a large number of these mutants affect the kinetics of recombination. They either reduce the efficiency of sister chromatid recombination or alter the assembly/disassembly of repair foci without affecting the products of recombination. These results suggest that the screen has identified a nu