title: The UvrD303 hyper-helicase exhibits increased processivity.
abstract: DNA helicases use energy derived from nucleoside 5'-triphosphate hydrolysis to catalyze the separation of double-stranded DNA into single-stranded intermediates for replication, recombination, and repair. Escherichia coli helicase II (UvrD) functions in methyl-directed mismatch repair, nucleotide excision repair, and homologous recombination. A previously discovered 2-amino acid substitution of residues 403 and 404 (both Asp → Ala) in the 2B subdomain of UvrD (uvrD303) confers an antimutator and UV-sensitive phenotype on cells expressing this allele. The purified protein exhibits a "hyper-helicase" unwinding activity in vitro. Using rapid quench, pre-steady state kinetic experiments we show the increased helicase activity of UvrD303 is due to an increase in the processivity of the unwinding reaction. We suggest that this mutation in the 2B subdomain results in a weakened interaction with the 1B subdomain, allowing the helicase to adopt a more open conformation. This is consistent with the idea that the 2B subdomain may have an autoregulatory role. The UvrD303 mutation may enable the helicase to unwind DNA via a "strand displacement" mechanism, which is similar to the mechanism used to processively translocate along single-stranded DNA, and the increased unwinding processivity may contribute directly to the antimutator phenotype.
authors.affiliation: From the Department of Biology.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Meiners
authors.affiliation: From the Department of Biology.
authors.forename: Kambiz
authors.initials: K
authors.lastname: Tahmaseb
authors.affiliation: From the Department of Biology, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 smatson@bio.unc.edu.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Matson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C506746:UvrD protein, E coli; D004265:DNA Helicases
keywords: ATPase; DNA Helicase; DNA Mismatch Repair; DNA Repair; Nucleotide Excision Repair; Processivity; Protein-Protein Interaction