The research in this laboratory focuses on a class of enzymes called helicases. These enzymes unwind duplex nucleic acids in an energy dependent reaction and play essential roles in all aspects of DNA metabolism in the cell. There are three major projects ongoing in the lab directed toward understanding these proteins. (1) An analysis, in enzymatic, molecular and genetic terms, of the mechanism of action and the physiological roles of four of the eleven helicases found in Escherichia coli. We are probing their reaction mechanisms and roles using biochemical, genetic and molecular biological techniques. This includes the construction of specific mutants by site-directed mutagenesis and the characterization of protein-DNA and protein-protein interactions. (2) Reconstitute the site and strand specific nicking and unwinding reaction required to initiate bacterial conjugation. Several genetic loci encoded on the bacterial F plasmid have been implicated in this fundamental reaction leading to DNA strand transfer. Three trans-acting proteins have been purified to homogeneity, and the nicking-unwinding reaction is being reconstituted through genetic and biochemical complementation studies. (3) Isolation and genetic/biochemical characterization of proteins with helicase activity from yeast. Several potential yeast helicase enzymes have been identified using both biochemcial and molecular biological methods. The biochemical characteristics of these enzymes and their roles in yeast nucleic acid metabolism will be evaluated.