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

A mutant allele of SGSI 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 Sgsl in the complementation of methylmethanesulfonate (MMS) sensitivity and the suppression of hyper recombination of a Sgsl mutant. In this study, we found that several amino acids residues in the N-terminal region of Sgsl between residues 4 and 33 were responsible for binding to Top3 and essential for complementing the sensitivity to MMS of Sgsl cells. Two-hybrid assays suggested that the region of Top3 responsible for the binding to Sgsl was bipartite, with portion in the N- and C-terminal domains. Although disruption of the SGSI gene suppressed the semi-lethality of the top3 mutant of strain MR, the sgsl-top3 double mutant grew more slowly and was more sensitive to MMS than the sgsl single mutant, indicating that Top3 plays some role independently of Sgsl. The DNA topoisomerase activ-ity 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 sgsl-top3 to the level of that of the sgsl single mutant. Epistatic analysis using the sgsl-top3 double mutant, rad52 mutant and sgsl-top3-rad52 triple mutant indicated that TOP3 belongs to the RAD52 recom-binational repair pathway.