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.
Sgs1, the RecQ helicase homolog, and Top3, the type-IA topoisomerase, physically interact and are required for genomic stability in budding yeast. Similarly, topoisomerase III genes physically pair with homologs of SGS1 in humans that are involved in the cancer predisposition and premature aging diseases Bloom, Werner, and Rothmund-Thompson syndromes. In the absence of Top1 activity, sgs1 mutants are severely growth impaired. Here, we investigate the role of Sgs1 helicase activity and its N-terminal Top3 interaction domain by using an allele replacement technique to integrate mutant alleles at the native SGS1 genomic locus. We compare the phenotype of helicase-defective (sgs1-hd) and N-terminal deletion (sgs1-NΔ) strains to wild-type and sgs1 null strains. Like the sgs1 null, sgs1-hd mutations suppress top3 slow growth, cause a growth defect in the absence of Srs2 helicase, and impair meiosis. However, for recombination and the synthetic interaction with top1Δ mutations, loss of helicase activity exhibits a less severe phenotype than the null. Interestingly, deletion of the Top3 interaction domain of Sgs1 causes a top3-like phenotype, and furthermore, this effect is dependent on helicase activity. These results suggest that the protein-protein interaction between these two DNA-metabolism enzymes, even in the absence of helicase activity, is important for their function in catalyzing specific changes in DNA topology.
In budding yeast, loss of topoisomerase III, encoded by the TOP3 gene, leads to a genomic instability phenotype that includes slow growth, hyper-sensitivity to genotoxic agents, mitotic hyper-recombination, increased chromosome missegregation, and meiotic failure. Slow growth and other defects of top3 mutants are suppressed by mutation of SGS1, which encodes the only RecQ helicase in S. cerevisiae. sgs1 is epistatic to top3, suggesting that the two proteins act in the same pathway. To identify other factors that function in the Sgs1-Top3 pathway, we undertook a genetic screen for non-sgs1 suppressors of top3 defects. We found that slow growth and DNA damage sensitivity of top3 mutants are suppressed by mutations in RAD51, RAD54, RAD55, and RAD57. In contrast, top3 mutants show extreme synergistic growth defects with mutations in RAD50, MRE11, XRS2, RDH54, and RAD1. We also analyzed recombination at the SUP4-o region, showing that in a rad51, rad54, rad55, or rad57 background top3Δ does not increase recombination to the same degree as in a wild-type strain. These results suggest that the presence of the Rad51 homologous recombination complex in a top3 background facilitates creation of detrimental intermediates by Sgs1. We present a model wherein Rad51 helps recruit Sgs1-Top3 to sites of replicative damage.
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