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Originally published In Press as doi:10.1074/jbc.M212916200 on February 20, 2003

J. Biol. Chem., Vol. 278, Issue 18, 16381-16388, May 2, 2003
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Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus*

Shelley L. Lusetti, Jeffrey J. Shaw, and Michael M. CoxDagger

From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mM Mg2+ in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg2+) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg2+ results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg2+ and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg2+ levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.

* This work was supported by National Institutes of Health Grant GM32335.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger 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@biochem.wisc.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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