DNA STRAND EXCHANGE PROMOTED BY RECA K72R - 2 REACTION PHASES WITH DIFFERENT MG2+ REQUIREMENTS

Replacement of lysine 72 in RecA protein with arginine produces a muta nt protein that binds but does not hydrolyze ATP. The protein neverthe less 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 concen tration 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 o bserved (albeit slowly). Higher levels of Mg2+ are required for optima l uptake of substrate duplex DNA into the nucleoprotein filament, indi cating that the formation of joint molecules is facilitated by Mg2+ le vels that inhibit the subsequent migration of a DNA branch. We also sh ow that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassin g structural barriers in the DNA or accommodating four DNA strands. Th e 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 co ntext 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 pro tein also facilitates the autocatalytic cleavage of the LexA repressor , but at a reduced rate.