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Single stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis
Tomko, Eric J. Fischer, Christopher J. Lohman, Timothy M.
Tomko, Eric J.
Fischer, Christopher J.
Lohman, Timothy M.
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Abstract
E. coli UvrD is an SF1A helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded (ss) DNA translocase, but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM) we proposed a non-uniform 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 nucleotides/step, suggesting a pause occurs every 4–5 nucleotides 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 nucleotides/step down to 50 μM ATP, but increases to ~7 nucleotides/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP, but with little futile ATP hydrolysis.
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Date
2012-02-14
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Elsevier
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Keywords
Helicase, Motor protein, Translocase, Kinetic step size, ATP coupling stoichiometry
Citation
Tomko, Eric J., Christopher J. Fischer, and Timothy M. Lohman. "Single-Stranded DNA Translocation of E. Coli UvrD Monomer Is Tightly Coupled to ATP Hydrolysis." Journal of Molecular Biology 418.1-2 (2012): 32-46.