Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme

Maria Spies; Mark S Dillingham; Stephen C Kowalczykowski

doi:10.1074/jbc.M505521200.

Back

Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme

Journal article

Open access

Peer reviewed

Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme

Maria Spies, Mark S Dillingham and Stephen C Kowalczykowski

The Journal of biological chemistry, Vol.280(44), pp.37078-37087

11/04/2005

DOI: 10.1074/jbc.M505521200.

PMID: 16041060

Files and links (1)

url

https://doi.org/10.1074/jbc.M505521200.View

Published (Version of record) Open Access

Abstract

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 chi and to coordinate the loading of RecA protein onto the ssDNA it produces. In contrast, the RecB(K29Q)CD 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(K29Q)CD enzyme displays helicase and nuclease activities, its inability to translocate along the 3'-terminated strand results in the failure to recognize chi and to load RecA protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to chi, whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.

Mutation

DNA Helicases - chemistry

Rec A Recombinases - metabolism

Mutagenesis, Site-Directed

DNA, Bacterial - metabolism

DNA, Single-Stranded - metabolism

Exodeoxyribonuclease V - chemistry

Adenosine Triphosphatases - metabolism

Escherichia coli Proteins - metabolism

Exodeoxyribonuclease V - metabolism

Protein Transport

Gene Expression Regulation, Enzymologic

Recombination, Genetic

Adenosine Triphosphate - metabolism

Rec A Recombinases - chemistry

Adenosine Triphosphatases - genetics

Gene Expression Regulation, Bacterial

Binding Sites

Details

Title: Subtitle: Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme
Creators: Maria Spies - Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616, USA
Mark S Dillingham
Stephen C Kowalczykowski
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.280(44), pp.37078-37087
Publisher: United States
DOI: 10.1074/jbc.M505521200.
PMID: 16041060
ISSN: 0021-9258
eISSN: 1083-351X
Grant note: GM-41347 / NIGMS NIH HHS
Language: English
Date published: 11/04/2005
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024557002771

Metrics

5 Record Views

34 Times Cited - Web of Science