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J Mol Biol. 2012 Apr 20;418(1-2):32-46. doi: 10.1016/j.jmb.2012.02.013. Epub 2012 Feb 14.

Single-stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis.

Author information

1
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110-1093, USA.

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.

PMID:
22342931
PMCID:
PMC3311787
DOI:
10.1016/j.jmb.2012.02.013
[Indexed for MEDLINE]
Free PMC Article

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