The genetic consequences of ablating helicase activity and the Top3 interaction domain of Sgs1

Abstract

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.

High-throughput creation and functional profiling of eukaryotic DNA sequence variant libraries using CRISPR/Cas9

“…Among the regions showing strongest depletion within edited cells were guide+donors deleting amino acid stretches 1-85, 686-1090 and 1116-1225 within Sgs1, which correspond to the Sgs1-Top3-binding domain, Sgs1-helicase, and RQC domains, respectively (2-tailed t-test, P<0.0001) (Figure 2, Supplementary File 1). These results are consistent with the known mechanism by which Sgs1 functions through the recruitment of accessory proteins (through N-terminal residues) [14][15][16][17][18][19][20][21][22] and by resolution of DNA structural intermediates via its helicase and RecQ domains 23,24 .…”

Pervasive and Essential Roles of the Top3-Rmi1 Decatenase Orchestrate Recombination and Facilitate Chromosome Segregation in Meiosis

“…To obtain evidence that direct interaction between Sgs1 and Top3 is important for regulating meiotic recombination, we also analyzed cells carrying the sgs1-ΔN82 allele, which lacks residues essential for interaction with Top3 and mimics the top3 mutant phenotype (Weinstein and Rothstein, 2008)(to circumvent slow growth caused by sgs1-ΔN82 , cells were made heterozygous with P CLB2 -SGS1 ). Meiotic phenotypes of sgs1-ΔN82 cells were indistinguishable from those conferred by P CLB2 alleles of the STR genes, including elevated ectopic recombination and altered timing of crossovers relative to noncrossovers (Figure S4).…”

The Dissolution of Double Holliday Junctions

“…In addition to this conserved genetic interaction, Sgs1/BLM RecQ helicases and type IA topoisomerases physically interact, as has been shown in S. cerevisiae between Top3 and Sgs1 (Bennett et al 2000;Fricke et al 2001), in S. pombe between Top3 and Rqh1 (Laursen et al 2003;Ahmad and Stewart 2005), and in human cells between BLM and TopoIIIa (Johnson et al 2000;Wu et al 2000). More important, this physical interaction is crucial for Sgs1/BLM to maintain genomic stability in vivo, because the first 100 aa residues of Sgs1, which mediate the physical association with Top3, are required for the complementation of the sgs1 phenotypes (Gangloff et al 1994;Bennett et al 2000;Duno et al 2000;Mullen et al 2000;Fricke et al 2001;Ui et al 2001;Onodera et al 2002;Ira et al 2003;Weinstein and Rothstein 2008). Similarly, the putative TopoIIIa interaction domain of BLM is necessary for the suppression of the elevated SCE phenotype of BS cells (Wu et al 2000;Hu et al 2001).…”