Abstract
Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg.dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg.dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.
Highlights
The RecA protein of Escherichia coli is a 352-amino acid polypeptide chain with a predicted molecular weight of 37,842
With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2؉ in ways that reflect the concentration of a Mg1⁄7dATP complex
We tested the capacity of the K72R mutant protein to promote unidirectional strand exchange, bypass of structural barriers in the DNA during strand exchange, and DNA strand exchange with four DNA strands, using strategies similar to those employed in several recent studies (Jain et al, 1994; Kim et al, 1992a, 1992b)
Summary
The RecA protein of Escherichia coli is a 352-amino acid polypeptide chain with a predicted molecular weight of 37,842. The reaction is apparently quite inefficient, with about 100 ATPs hydrolyzed per base pair of hybrid DNA created The function of this ATP hydrolysis is incompletely understood. An important clue was provided by the observation that RecA protein can promote DNA strand exchange under some conditions in the presence of ATP␥S, an ATP analog that is not readily hydrolyzed by RecA (Menetski et al, 1990; Rosselli and Stasiak, 1990). This observation was reinforced more recently by work on the RecA mutant K72R (Rehrauer and Kowalczykowski, 1993) and again with wild type RecA protein in the presence of ADP-AlF4Ϫ (Kowalczykowski and Krupp, 1995). A series of studies determined that the strand exchange occurring with ATP␥S is limited in extent and bidirectional
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