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J. Biol. Chem., Vol. 280, Issue 44, 37069-37077, November 4, 2005

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Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme^*

Mark S. Dillingham1, Martin R. Webb, and Stephen C. Kowalczykowski2

From the Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616 and the National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is 50%. More significantly, the processivity of translocation is substantially reduced by 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.

Received for publication, May 20, 2005 , and in revised form, July 22, 2005.

^* This work was supported by a Wellcome Trust Traveling Research Fellowship (to M. S. D.), by the Medical Research Council, and by National Institutes of Health Grant GM-41347 (to S. C. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Current address: University of Bristol, DNA-protein Interactions Group, Dept. of Biochemistry, School of Medical Sciences, Bristol BS8 1TD, UK.

² To whom correspondence should be addressed: University of California, Davis, Section of Microbiology, Center for Genetics and Development, One Shields Ave., Briggs Hall 310, Davis, CA 95616-8665. Tel.: 530-752-5938; Fax: 530-752-5939; E-mail: sckowalczykowski{at}ucdavis.edu.

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