title: Kinetic mechanism for formation of the active, dimeric UvrD helicase-DNA complex.
abstract: Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Nasib K
authors.initials: NK
authors.lastname: Maluf
authors.forename: Janid A
authors.initials: JA
authors.lastname: Ali
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins; D007700:Kinetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases