title: Single-molecule assay reveals strand switching and enhanced processivity of UvrD.
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.
authors.affiliation: Laboratoire de Physique Statistique, Ecole Normale Supérieure, Unité Mixte de Recherche 8550, Centre National de la Recherche Scientifique, 24 Rue Lhomond, 75231 Paris Cedex 05, France.
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Dessinges
authors.forename: Timothée
authors.initials: T
authors.lastname: Lionnet
authors.forename: Xu Guang
authors.initials: XG
authors.lastname: Xi
authors.forename: David
authors.initials: D
authors.lastname: Bensimon
authors.forename: Vincent
authors.initials: V
authors.lastname: Croquette
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000737:chemistry*; D007700:Kinetics; D013379:Substrate Specificity; D013698:Templates, Genetic
chemical_list: D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases