Escherichia coli DNA helicase II: Structure-function analysis of conserved amino acid motifs and investigation of protein-protein interaction

Helicases catalyze the unwinding of duplex nucleic acids using energy derived from nucleoside triphosphate hydrolysis. They are essential components of the enzymatic machinery responsible for metabolizing DNA and RNA in all living organisms. Helicases from viral, prokaryotic, and eukaryotic systems share regions of amino acid sequence homology and it has been hypothesized that these conserved "motifs" are critical for enzymatic function.;I examined the functional significance of two conserved amino acid motifs in Escherichia coli DNA helicase II (UvrD), a member of helicase superfamily 1. The most highly conserved residues in motifs IV and VI were altered by site-directed mutagenesis. Genetic and biochemical assays were used to compare mutant and wild-type proteins to measure the effects of the mutations on the in vivo function and in vitro biochemical properties of UvrD. The results indicate that motif IV is involved in ATP binding and that motif VI is important for conformational changes in the enzyme used to couple the ATP hydrolysis and ssDNA binding activities.;I also used a yeast two-hybrid screen to search for UvrD-interacting proteins from E. coli that would further our understanding of the roles of UvrD in DNA metabolism. The screen revealed that UvrD interacts with itself and with MutL, a protein involved with UvrD in DNA mismatch repair. Affinity chromatography was used to confirm the MutL-UvrD interaction. In addition, MutL greatly stimulated the helicase, but not the ATPase, activity of UvrD. Mismatch repair is responsible for correcting replication errors in E. coli. A homologous pathway exists in humans and has been linked to cancer. The implications of the MutL-UvrD interaction for the mismatch repair mechanism in E. coli and humans are explored.;Biophysical and biochemical experiments using UvrD and a UvrD mutant that fails to interact with itself suggested that the oligomerization of UvrD is not required for its activity as a helicase. Furthermore, inactive UvrD point mutants did not inhibit the ATPase or helicase activities of UvrD. An active monomeric helicase has not been described previously and I present a model for duplex DNA unwinding by a monomeric enzyme.