A nonuniform stepping mechanism for E. coli UvrD monomer translocation along single-stranded DNA.
E. coli UvrD is an SF1 helicase involved in several DNA metabolic processes. Although a UvrD dimer is needed for helicase activity, a monomer can translocate with 3' to 5' directionality along single-stranded DNA, and this ATP-dependent translocation is likely involved in RecA displacement. In order to understand how the monomeric translocase functions, we have combined fluorescence stopped-flow kinetic methods with recently developed analysis methods to determine the kinetic mechanism, including ATP coupling stoichiometry, for UvrD monomer translocation along ssDNA. Our results suggest that the macroscopic rate of UvrD monomer translocation is not limited by each ATPase cycle but rather by a slow step (pause) in each translocation cycle that occurs after four to five rapid 1 nt translocation steps, with each rapid step coupled to hydrolysis of one ATP. These results suggest a nonuniform stepping mechanism that differs from either a Brownian motor or previous structure-based inchworm mechanisms.
Tomko
Eric J
EJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110-1093, USA.
Fischer
Christopher J
CJ
Niedziela-Majka
Anita
A
Lohman
Timothy M
TM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002232:Carbocyanines; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D019793:Fluorescein; D007700:Kinetics; D008954:Models, Biological; D053484:RecQ Helicases / Q000378:metabolism*; D014178:Translocation, Genetic / Q000502:physiology*
D002232:Carbocyanines; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C089187:cyanine dye 3; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases; D019793:Fluorescein