Academic journal article Genetics

A Genetic Screen for Top3 Suppressors in Saccharomyces Cerevisiae Identifies SHU1, SHU2, PSY3 and CSM2: Four Genes Involved in Error-Free DNA Repair

Academic journal article Genetics

A Genetic Screen for Top3 Suppressors in Saccharomyces Cerevisiae Identifies SHU1, SHU2, PSY3 and CSM2: Four Genes Involved in Error-Free DNA Repair

Article excerpt

ABSTRACT

Helicases of the RecQ family and topoisomerase III are evolutionarily conserved proteins important for maintenance of genome stability. In Saccharomyces cerevisiae, loss of the TOP3 gene, encoding topoisomerase III, results in a phenotype of slow growth, DNA damage sensitivity, meiotic defects, and hyperrecombination. The sole RecQ helicase in budding yeast, Sgs1, interacts with Top3 both physically and genetically, and the two proteins are thought to act in concert in vivo. Much recent genetic and biochemical evidence points to the role of RecQ helicases and topoisomerase III in regulating homologous recombination (HR) during DNA replication. Previously, we found that mutations in HR genes partially suppress top3 slow growth. Here, we describe the analysis of four additional mutational suppressors of top3 defects: shu1, shu2, psy3, and csm2. These genes belong to one epistasis group and their protein products interact with each other, strongly suggesting that they function as a complex in vivo. Their mutant phenotype indicates that they are important for error-free repair of spontaneous and induced DNA lesions, protecting the genome from mutation. These mutants exhibit an epistatic relationship with rad52 and show altered dynamics of Rad52-YFP foci, suggesting a role for these proteins in recombinational repair.

ALL living cells possess mechanisms that ensure faithful reproduction of genetic material and protection of the genome from excessive mutation. Mutagenic DNA lesions can either arise spontaneously, for instance, during the process of DNA replication, or be induced by exogenous agents, such as chemicals or irradiation. One of the pathways used to repair both spontaneous and induced DNA double-strand breaks (DSBs) or single-strand gaps is homologous recombination (HR), a process of exchange of genetic material between two DNA molecules of nearly or perfectly identical sequence (SYMINGTON 2002). Since it uses a homologous sequence as template for DNA repair, normal HR is error free, thus playing an important role in protection of the genome from mutation. However, recombination has to be regulated, since excessive or inappropriate recombination can also contribute to genomic instability. A growing amount of evidence suggests that helicases of the RecQ family in conjunction with type III topoisomerases play crucial roles in regulating HR (UAKLEY and HICKSON 2002; KHAKHAR et al 2003).

Topoisomerase III was first identified in budding yeast as a mutation that causes a hyperrecombination phenotype at the SUP4-O locus (WALLIS et al. 1989). In addition to causing increased recombination, mutation of TOP3 results in slow growth, hypersensitivity to genotoxic agents, such as hydroxyurea (HU) and methyl methanesulfonate (MMS), mitotic chromosome missegregation, and inability to undergo meiosis and sporulate (WALLIS et al. 1989; GANGLOFF et al. 1999; CHAKRAVERTY et al. 2001). Mutation of SGSl, the gene encoding the sole budding yeast RecQ helicase, suppresses top3L· slow growth and the two proteins physically interact with each other (GANGLOFF et al. 1994; BENNETT et al. 2000; FRICKE et al. 2001). Mutation of SGSl in an otherwise wild-type background causes a phenotype reminiscent of that of the top3A strain, although milder in severity (GANGLOFF et al. 1994; WATT et al. 1995, 1996).

RecQ helicases and topoisomerase III are evolutionarily conserved proteins and play important roles in maintenance of genome stability in higher eukaryotes. For instance, mutations in three of five human RecQ homologs, BLM, WRN, and RECQL4, cause the cancer predisposition syndromes Bloom, Werner, and Rothmund-Thomson (reviewed in HICKSON 2003). Werner syndrome and Rothmund-Thomson syndrome patients also exhibit symptoms of premature aging. At the cellular level, all of these syndromes are characterized by genomic instability that is, at least in part, caused by inappropriate recombination events. The functional interaction between RecQ-like helicases and topoisomerase III homologs also appears to be conserved in human cells. …

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