Single-molecule assay reveals strand switching and enhanced processivity of UvrD

Publication Type:

Journal Article

Source:

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, NATL ACAD SCIENCES, Volume 101, Number 17, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, p.6439-6444 (2004)

DOI:

10.1073/pnas.0306713101

Keywords:

DNA HELICASE II; ESCHERICHIA-COLI-UVRD; GENE; INITIATION; MACROMOLECULAR MACHINES; MECHANISMS; MOVEMENT; PROTEIN; REPLICATION; TRANSLOCATION

Abstract:

DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3'-5' on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.