The Bloom's syndrome DNA helicase complex: Identification and characterization of activities conserved in the orthologous complex from Saccharomyces cerevisiae

loom's Syndrome (BS) is a rare human disease characterized by genome instability and cancer predisposition. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. This family consists of five human paralogs that play crucial roles in guarding against DNA rearrangements. All BLM orthologs, including budding yeast Sgs1, bind stably to a protein complex composed of DNA topoisomerase 3alpha (Top3) and the OB-fold protein Rmi1. Although the BLM/Sgs1 complex is known to suppress homologous recombination, its mechanism of action is unknown.;I found that a stable Top3-Rmi1 complex can be isolated from yeast cells overexpressing these two subunits and it shows increased superhelical relaxation activity compared to Top3 alone. The Rmi1 subunit also stimulates Top3 activity in reconstitution experiments. In both cases, elevated temperatures are required for optimal relaxation unless the substrate contains a ssDNA bubble. Interestingly, Rmi1 binds only weakly to ssDNA on its own, but it stimulates the ssDNA binding activity of Top3 five-fold. Top3 and Rmi1 also cooperate to bind the Sgs1 N-terminus and promote its interaction with single-strand (ss) DNA.;In addition to the highly-conserved DNA helicase domain, all BLM/Sgs1 orthologs contain a large (652 aa) N-terminal domain that has no known catalytic activity. To determine the function of the N-terminal domain, I assayed truncated Sgs1 proteins for ssDNA binding activity. I identified a sub-domain of the Sgs1 N-terminus (SE, aa...