Abstract
BackgroundIn Saccharomyces cerevisiae, Rad59 is required for multiple homologous recombination mechanisms and viability in DNA replication-defective rad27 mutant cells. Recently, four rad59 missense alleles were found to have distinct effects on homologous recombination that are consistent with separation-of-function mutations. The rad59-K166A allele alters an amino acid in a conserved α-helical domain, and, like the rad59 null allele diminishes association of Rad52 with double-strand breaks. The rad59-K174A and rad59-F180A alleles alter amino acids in the same domain and have genetically similar effects on homologous recombination. The rad59-Y92A allele alters a conserved amino acid in a separate domain, has genetically distinct effects on homologous recombination, and does not diminish association of Rad52 with double-strand breaks.ResultsIn this study, rad59 mutant strains were crossed with a rad27 null mutant to examine the effects of the rad59 alleles on the link between viability, growth and the stimulation of homologous recombination in replication-defective cells. Like the rad59 null allele, rad59-K166A was synthetically lethal in combination with rad27. The rad59-K174A and rad59-F180A alleles were not synthetically lethal in combination with rad27, had effects on growth that coincided with decreased ectopic gene conversion, but did not affect mutation, unequal sister-chromatid recombination, or loss of heterozygosity. The rad59-Y92A allele was not synthetically lethal when combined with rad27, stimulated ectopic gene conversion and heteroallelic recombination independently from rad27, and was mutually epistatic with srs2. Unlike rad27, the stimulatory effect of rad59-Y92A on homologous recombination was not accompanied by effects on growth rate, cell cycle distribution, mutation, unequal sister-chromatid recombination, or loss of heterozygosity.ConclusionsThe synthetic lethality conferred by rad59 null and rad59-K166A alleles correlates with their inhibitory effect on association of Rad52 with double-strand breaks, suggesting that this may be essential for rescuing replication lesions in rad27 mutant cells. The rad59-K174A and rad59-F180A alleles may fractionally reduce this same function, which proportionally reduced repair of replication lesions by homologous recombination and growth rate. In contrast, rad59-Y92A stimulates homologous recombination, perhaps by affecting association of replication lesions with the Rad51 recombinase. This suggests that Rad59 influences the rescue of replication lesions by multiple recombination factors.
Highlights
In Saccharomyces cerevisiae, Rad59 is required for multiple homologous recombination mechanisms and viability in DNA replication-defective rad27 mutant cells
The rad59 mutant alleles display distinct effects on survival and growth in cells defective for lagging strand synthesis To further explore the function of RAD59 required for viability in rad27 null mutant cells, the effects of combining the rad27::LEU2 allele with the various rad59 alleles were determined by examining their ability to yield viable spores upon co-segregation in genetic crosses
The rad59-K166A allele, which alters a conserved lysine in the region of Rad59 that corresponds to the α-helical domain of the β − β − β − α motif of human Rad52 (Additional file 1: Figure S1) [27,34,35] displayed the same failure to appear with the rad27::LEU2 allele, indicative of synthetic lethality
Summary
In Saccharomyces cerevisiae, Rad is required for multiple homologous recombination mechanisms and viability in DNA replication-defective rad mutant cells. The rad59-K166A allele alters an amino acid in a conserved α-helical domain, and, like the rad null allele diminishes association of Rad with double-strand breaks. The rad59-Y92A allele alters a conserved amino acid in a separate domain, has genetically distinct effects on homologous recombination, and does not diminish association of Rad with double-strand breaks. Concomitant loss of Rad and components of the HR apparatus leads to synthetic lethality [18,19,20] These observations implicate HR in repair of DSBs that accumulate in the absence of Rad. Failure to repair DSBs leads to chromosome loss [21] that is greatly stimulated in rad null mutant cells [8], suggesting that the essential role for the HR apparatus in rad mutants may be prevention of lethal levels of chromosome loss
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