Functional and physical interaction between Sgs1 and Top3 and Sgs1-independent function of Top3 in DNA recombination repair.

Abstract

A mutant allele of SGS1 of Saccharomyces cerevisiae was identified as a suppressor of the slow-growth phenotype of top3 mutants. We previously reported the involvement of Top3 via the interaction with the N-terminal region of Sgs1 in the complementation of methylmethanesulfonate (MMS) sensitivity and the suppression of hyper recombination of a sgs1 mutant. In this study, we found that several amino acids residues in the N-terminal region of Sgs1 between residues 4 and 33 were responsible for binding to Top3 and essential for complementing the sensitivity to MMS of sgs1 cells. Two-hybrid assays suggested that the region of Top3 responsible for the binding to Sgs1 was bipartite, with portion in the N-and C-terminal domains. Although disruption of the SGS1 gene suppressed the semilethality of the top3 mutant of strain MR, the sgs1-top3 double mutant grew more slowly and was more sensitive to MMS than the sgs1 single mutant, indicating that Top3 plays some role independently of Sgs1. The DNA topoisomerase activity of Top3 was required for the Top3 function to repair DNA damages induced by MMS, as shown by the fact that the TOP3 gene carrying a mutation (Phe for Tyr) at the amino acid residue essential for its activity (residue 356) failed to restore the MMS sensitivity of sgs1-top3 to the level of that of the sgs1 single mutant. Epistatic analysis using the sgs1-top3 double mutant, rad52 mutant and sgs1-top3-rad52 triple mutant indicated that TOP3 belongs to the RAD52 recombinational repair pathway.

Role for the Fission Yeast RecQ Helicase in DNA Repair in G2

“…In other words, the rescue of the growth defect of rqh1-d rad22-d by deletion of top3 is evidence that Top3 functions independently of Rqh1 but not that Rqh1 functions independently of Top3. This is consistent with work by Onodera et al (43), who reported a functional and physical interaction between Sgs1 and Top3 and an Sgs1-independent function of Top3 in DNA recombination repair (43). In all our experiments we have not been able to separate Rqh1 function from Top3 function.…”

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 .…”

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

“…In most of these reports, alleles expressed from plasmids or integrated at non-native loci were used to study complementation [40,41,45,46,52,[64][65][66][67][68]. To avoid the ambiguities inherent in interpreting growth or lack of growth in cells transformed with a plasmid as well as to avoid potential position effects for loci integrated elsewhere, all mutant alleles used in this study were integrated at the SGS1 genomic locus (Table 1) [53,54].…”