Single-Stranded DNA Translocation of E. coli UvrD Monomer Is Tightly Coupled to ATP Hydrolysis

JOURNAL OF MOLECULAR BIOLOGY

卷:418

Article

Tomko, Eric J.¹  Fischer, Christopher J.²  Lohman, Timothy M.¹ 

[1] Washington Univ, Sch Med, Dept Biochem & Mol Biophys, St Louis, MO 63110 USA

[2] Univ Kansas, Dept Phys & Astron, Lawrence, KS 66049 USA

关键词: helicase;    motor protein;    translocase;    kinetic step size;    ATP coupling stoichiometry;   

DOI  :  10.1016/j.jmb.2012.02.013

来源: Elsevier

【 摘 要 】

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 mu M), 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 mu M ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of similar to 1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 mu M), 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 mu M ATP but increases to similar to 7 nt/step at 10 mu M ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP but with little futile ATP hydrolysis. (C) 2012 Elsevier Ltd. All rights reserved.

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