HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 44, 37078-37087, November 4, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/44/37078    most recent M505521200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Spies, M. Articles by Kowalczykowski, S. C. Search for Related Content PubMed PubMed Citation Articles by Spies, M. Articles by Kowalczykowski, S. C. Social Bookmarking                      What's this?

Translocation by the RecB Motor Is an Absolute Requirement for -Recognition and RecA Protein Loading by RecBCD Enzyme^*

From the Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616

RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. The enzyme is driven by two motor subunits, RecB and RecD, translocating on opposite single-strands of the DNA duplex. Here we provide evidence that, although both motor subunits can support the translocation activity for the enzyme, the activity of the RecB subunit is necessary for proper function of the enzyme both in vivo and in vitro. We demonstrate that the RecBCD^K177Q enzyme, in which RecD helicase is disabled by mutation of the ATPase active site, complements recBCD deletion in vivo and displays all of the enzymatic activities that are characteristic of the wild-type enzyme in vitro. These include helicase and nuclease activities and the abilities to recognize the recombination hotspot and to coordinate the loading of RecA protein onto the ssDNA it produces. In contrast, the RecB^K29QCD enzyme, carrying a mutation in the ATPase site of RecB helicase, fails to complement recBCD deletion in vivo. We further show that even though RecB^K29QCD enzyme displays helicase and nuclease activities, its inability to translocate along the 3'-terminated strand results in the failure to recognize and to load RecA protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to , whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.

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

^* This work was supported by National Institute of Health Grant GM-41347 (to S. C. K.), by American Cancer Society Postdoctoral Fellowship PF-02-116-01-GMC (to M. S.), and by Wellcome Trust Traveling Research Fellowship (M. S. D.). 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.

¹ Current address: University of Bristol, DNA-protein Interactions Group, Department 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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. S. Dillingham and S. C. Kowalczykowski RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks Microbiol. Mol. Biol. Rev., December 1, 2008; 72(4): 642 - 671. [Abstract] [Full Text] [PDF]

				S. K. Amundsen, A. F. Taylor, M. Reddy, and G. R. Smith Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots Genes & Dev., December 15, 2007; 21(24): 3296 - 3307. [Abstract] [Full Text] [PDF]

				M. S. Dillingham, M. R. Webb, and S. C. Kowalczykowski Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme J. Biol. Chem., November 4, 2005; 280(44): 37069 - 37077. [Abstract] [Full Text] [PDF]