Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli

Naofumi Handa; Katsumi Morimatsu; Susan T. Lovett; Stephen C. Kowalczykowski

doi:10.1101/gad.1780709

Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli

¹Department of Microbiology, University of California at Davis, Davis, Calfironia 95616, USA;
²Department of Molecular and Cellular Biology, University of California at Davis, Davis, Calfironia 95616, USA;
³Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, Massachusetts 02454, USA

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↵4 These authors contributed equally to this work.

Abstract

The RecF pathway of Escherichia coli is important for recombinational repair of DNA breaks and gaps. Here `we reconstitute in vitro a seven-protein reaction that recapitulates early steps of dsDNA break repair using purified RecA, RecF, RecO, RecR, RecQ, RecJ, and SSB proteins, components of the RecF system. Their combined action results in processing of linear dsDNA and its homologous pairing with supercoiled DNA. RecA, RecO, RecR, and RecJ are essential for joint molecule formation, whereas SSB and RecF are stimulatory. This reconstituted system reveals an unexpected essential function for RecJ exonuclease: the capability to resect duplex DNA. RecQ helicase stimulates this processing, but also disrupts joint molecules. RecO and RecR have two indispensable functions: They mediate exchange of RecA for SSB to form the RecA nucleoprotein filament, and act with RecF to load RecA onto the SSB–ssDNA complex at processed ssDNA–dsDNA junctions. The RecF pathway has many parallels with recombinational repair in eukaryotes.

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Footnotes

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↵5 Present address: Laboratory of Social Genome Sciences, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
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↵6 Corresponding author.

↵E-MAIL sckowalczykowski{at}ucdavis.edu; FAX (530) 752-5939.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1780709.
Supplemental material is available at http://www.genesdev.org.
- Received January 12, 2009.
- Accepted April 8, 2009.