title: A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro.
abstract: The Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized in vitro UvrD-catalyzed unwinding of a series of 18 bp duplex DNA substrates with 3' single-stranded DNA (ssDNA) tails ranging in length from two to 40 nt. Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both [UvrD] and [DNA] under conditions such that UvrD-DNA binding is stoichiometric. Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3' ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths </=8 nt, even at high [UvrD]/[DNA] ratios. Unwinding is observed for DNA substrates with 3' ssDNA tail lengths >/=12 nt, and the unwinding amplitude displays a sigmoidal dependence on [UvrD(tot)]/[DNA(tot)]. Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3' ssDNA tail length, is not competent to fully unwind even a short 18 bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro. Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E.coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP. Initiation of DNA unwinding in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3' ssDNA tail.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8231, St. Louis, MO 63110-1093, USA.
authors.forename: Nasib K
authors.initials: NK
authors.lastname: Maluf
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fischer
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001667:Binding, Competitive; D002213:Capsid / Q000502:physiology; D036022:Capsid Proteins; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D066298:In Vitro Techniques; D007700:Kinetics; D008433:Mathematics; D011485:Protein Binding; D013696:Temperature; D014461:Ultracentrifugation
chemical_list: D036022:Capsid Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C506744:rep protein, E coli; C061379:gp20 protein, bacteriophage T4; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases