The Homologous Recombination Gene, RAD59, is a Determinant of Genome Stability in Saccharomyces cerevisiae

Rad59 is a DNA repair protein involved in double-strand break repair (DSBR) by homologous recombination (HR) in Saccharomyces cerevisiae. Rad59 aids the central HR protein, Rad52, in multiple mechanisms of HR. Rad59 shares sequence homology with the N terminus of Rad52. We previously made mutations in highly conserved residues in a conserved domain between Rad59 and Rad52, including rad59-Y92A, rad59-K166A, rad59-K174A, and rad59-F180A. The rad59-K166A, rad59-K174A, and rad59-F180A mutations reside within the same &agr;-helical domain that is thought to be important for DNA binding. The rad59-Y92A mutation resides in a separate loop domain. Co-immunoprecipitation (Co-IP) studies established that Rad59 works with Rad52 through their direct interaction. The Rad59-Y92A and Rad59-K166A mutant proteins disturb their physical interaction with Rad52. Chromatin immunoprecipitation (ChIP) analysis revealed that flag-tagged Rad52 associates with induced DSBs. This association is inhibited in the presence of the rad59-null and rad59-K166A mutant alleles, but is stimulated in the presence of rad59-Y92A. Epistasis analysis revealed that the rad59-K166A and rad59-Y92A mutations confer distinct genetic effects on single-strand annealing (SSA), a non-conservative mechanism of HR. Taken together, these findings indicate that the rad59 mutations have distinct effects that are consistent with the separation-of-function of Rad59 in HR.;Rad59 has also been implicated in the recovery of telomerase-deficient yeast cells from the viability loss associated with senescence. Senescence is the point at which cells stop dividing. This process is associated with the gradual loss of chromosome ends, called the telomere. Some human cells, including germ and stem cells (and wild-type yeast cells), express an enzyme called telomerase that extends telomere ends. Telomerase is inactivated in the majority of somatic cells, however, leaving them vulnerable to telomere loss and senescence. This makes telomerase-deficient yeast cells an excellent system to examine processes involved in telomere loss and senescence. Rad52-dependent HR is required for cells to rebuild their telomeres and recover from senescence. Rad59 aids Rad52 in this process by one mechanism of HR (Type II).;Nearly two decades have passed since the discovery of Rad59, yet the mechanism for how Rad59 works with Rad52 in multiple contexts of HR remains largely unknown. Therefore, this work aimed to better define the mechanism of Rad59 in HR in response to DSBs induced in two distinct, biologically relevant, contexts: 1) in response to spontaneous replication lesions in cells defective for lagging strand synthesis, and 2) in response to the lesions associated with gradual telomere loss during senescence.;To address the role of Rad59 in the HR-dependent recovery of cells from spontaneous replication failure, 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..;To address the role of Rad59 in the HR-dependent recovery of telomerase-deficient cells from senescence, mutant strains containing the rad59 &agr;-helical domain mutations were crossed with est2-null cells defective for telomerase. The kinetics of the descent into and recovery from senescence were then monitored in liquid culture. Southern blot analysis was subsequently used to measure the recombination-dependent recovery of telomeres. The est2Deltarad59-double mutant cells displayed a severity gradient in both the descent into senescence and recovery of telomeres by HR that mirrored those in response to spontaneous replication failure: rad59-K166A was the most defective, rad59-F180A was the next most defective, and rad59-K174A was the least defective of the mutations. This severity defect was consistent with the pattern of telomere recovery observed in Southern blots: est2Deltarad59-K166A cells recovered by the same mechanism as est2Deltarad59Delta cells with no functional Rad59 (Type I), whereas the est2Deltarad59-K174A and est2Deltarad59-Y92A cells recovered by another mechanism that requires Rad59 (Type II). Genetic analysis of the est2Deltarad51Deltarad59-triple mutant cells revealed that RAD59 works with RAD51 in the recovery of telomeres by HR.;These studies were the first to employ rad59 mutations to directly investigate the ability of cells to recover from spontaneous replication failure by HR. The data gathered directly implicates RAD59-dependent HR, and not just the HR apparatus, in the repair of DNA lesions that block growth. Additionally, epistasis analysis provided evidence that RAD59 works with RAD51 and SRS2 to mediate the recombination-dependent repair of spontaneous replication lesions. These data establish that RAD59 plays a role in promoting and limiting HR driven by replication failure, suggesting that both are required to facilitate growth and limit genome instability. Furthermore, these were the first studies to use rad59 mutations to investigate the role of RAD59 in the survival of telomerase-deficient cells from senescence-associated viability loss. The data gathered demonstrate, for the first time, that RAD59 works with RAD51 in the recovery of telomeres by HR. Taken together, these findings significantly enhance our understanding of the role the HR factor, Rad59, plays in the maintenance of genome stability in multiple biological contexts.