Disassembly of Escherichia coli RecA E38K/ΔC17 Nucleoprotein Filaments Is Required to Complete DNA Strand Exchange*
- Rachel L. Britt‡,
- Nami Haruta§,
- Shelley L. Lusetti¶,
- Sindhu Chitteni-Pattu‡,
- Ross B. Inman‡ and
- Michael M. Cox‡,1
- From the ‡Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706,
- the §Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan, and
- the ¶Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
- 1 To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706-1544. Tel.: 608-262-1181; Fax: 608-265-2603; E-mail: cox{at}biochem.wisc.edu.
Abstract
Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/ΔC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/ΔC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/ΔC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.
Footnotes
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↵* This work was supported, in whole or in part, by National Institutes of Health Grant GM32335 (to M. M. C.).
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This paper is dedicated to the memory of our longtime friend and colleague, Ross B. Inman.
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↵3 R. Britt, unpublished observations.
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↵2 The abbreviations used are:
- ssDNA
- single-stranded DNA
- DTT
- dithiothreitol
- dsDNA
- double-stranded DNA
- CHES
- 2-(cyclohexylamino)ethanesulfonic acid
- MES
- 4-morpholineethanesulfonic acid
- ATPγS
- adenosine 5′-O-(thiotriphosphate)
- SSB
- single strand DNA-binding protein
- ldsDNA
- linear double-stranded DNA
- cssDNA
- circular ssDNA.
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- Received June 2, 2009.
- Revision received October 16, 2009.
- © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
