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 ?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 ?M 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 ?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 ?M ATP but increases to ?7 nt/step at 10 ?M ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP but with little futile ATP hydrolysis.

SUBMITTER: Tomko EJ 

PROVIDER: S-EPMC3311787 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature