title: Single-stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis.
abstract: Escherichia coli UvrD is an SF1A (superfamily 1 type A) helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded DNA (ssDNA) translocase but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM), we proposed a nonuniform stepping mechanism in which a UvrD monomer translocates with biased (3' to 5') directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step size of 4-5 nt/step, suggesting that a pause occurs every 4-5 nt translocated. To further test this mechanism, we examined UvrD translocation over a range of lower ATP concentrations (10-500 μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ∼1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 μM), indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4-5 nt/step down to 50 μM ATP but increases to ∼7 nt/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP but with little futile ATP hydrolysis.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110-1093, USA.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Tomko
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fischer
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Lohman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006868:Hydrolysis; D021381:Protein Transport
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; C506746:UvrD protein, E coli; D004265:DNA Helicases