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.helicase ͉ DNA replication ͉ DNA repair ͉ magnetic tweezers A lthough helicases are essential molecular motors, their precise mechanism is only partially known. These motors translocate along DNA while stripping off one strand of the double helix (1, 2). Whereas a large number of helicases involved in DNA repair and recombination are either monomeric or dimeric, replicative helicases typically form processive, hexameric entities surrounding one or both strands. The process of separating the two strands of DNA is often described as the translocation of the enzyme on one strand, which defines the directionality of the process, whereas the displacement of the other strand is accomplished actively, if the helicase melts the base pairs, or, passively, if the helicase moves forward as the bases transiently unpair. Observing the activity of a single helicase unwinding a double helix yields valuable information on the enzymatic dynamics, as has been the case for other molecular motors such as kinesin and myosin (3).UvrD (720 aa, molecular mass ϭ 82 kDa), a member of the helicase SF1 superfamily (which includes PcrA and Rep), plays a crucial role in nucleotide excision repair and methyl-directed mismatch repair (4-8) and is required for the replication of several plasmids (9). It has been shown to initiate unwinding from a 3Ј end single-stranded DNA (ssDNA) tail, a gap, or a nick and to translocate along ssDNA in a 3Ј-5Ј direction (10-12). The purpose of this study is to investigate the mechanochemistry of UvrD-catalyzed DNA unwinding at the single-molecule level, yielding more direct insight into its enzymatic activity by avoiding the inherent averaging of bulk assays. We present real-time measurements of the unwinding rate, lifetime, and number of base pairs unwound, as well as an estimate of the step size. In addition, we observe that an unwinding event can be followed by an enzyme-translocation-limited rehybridization of the o...

Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvA–RuvB protein complex

“…have been analyzed by single-molecule techniques (20)(21)(22)(23). RecBCD and UvrD are highly processive helicases, and furthermore, RecBCD is a highly processive nuclease, hence their motion could be visualized (20,21,23). Rep was analyzed by a refined single-molecule FRET technique (22).…”

DNA Helicase Activity of the RecD Protein from Deinococcus radiodurans

“…Greater unwinding rates might be observed either at higher DNA concentrations (V max conditions), or at higher protein concentrations under single turnover conditions. Some helicases do unwind DNA much more rapidly than does RecD, including RecBCD (38) and E. coli UvrD (39).…”

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

“…E. coli UvrD shows specificity for binding 5Ј-ss/dsDNA junctions (40) and is capable of switching strands during translocation upon encountering a DNA fork (41). E. coli RecBC has been reported to consist of primary and secondary translocase activities that facilitate its translocation along both strands of a duplex simultaneously (42,43).…”