Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase

Several members of the RecQ family of DNA helicases are known to interact w ith DNA topoisomerase III (Top3). Here we show that the Saccharomyces cerev isiae Sgs1 and Top3 proteins physically interact in cell extracts and bind directly in vitro. Sgs1 and Top3 proteins coimmunoprecipitate from cell ext racts under stringent conditions, indicating that Sgs1 and Top3 are present in a stable complex, The domain of Sgs1 which interacts with Top3 was iden tified by expressing Sgs1 truncations in yeast. The results indicate that t he NH2-terminal 158 amino acids of Sgs1 are sufficient for the high affinit y interaction between Sgs1 and Top3. lit vitro assays using purified Top3 a nd NH2-terminal Sgs1 fragments demonstrate that at feast part of the intera ction is through direct protein-protein interactions with these 158 amino a cids. Consistent with these physical data, we find that mutant phenotypes c aused by a point mutation or small deletions in the Sgs1 NH2 terminus can b e suppressed by Top3 overexpression. We conclude that Sgs1 and Top3 form a tight complex in vivo and that the first 158 amino acids of Sgs1 are necess ary and sufficient for this interaction. Thus, a primary role of the Sgs1. amino terminus is to mediate the Top3 interaction.