Analysis of the functional domains of a regulatory protein, RecA, using gene manipulation and X-ray crystallography.

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For its size (38,000 daltons), the RecA protein exhibits a remarkable variety of reactions in vitro: RecA protein, ( <i> ) binds to a single-stranded DNA; ( <ii> ) has DNAdependent ATPase; ( <iii> ) catalyzes the renaturation of complementary single-strands of DNA; ( <iv> ) unwinds double stranded DNA; and ( <v> ) cleaves LexA protein and the repressors of <lambda> , P22, and <phi> 80 phage. RecA protein probably has several different sites involved directly in these various reactions. To elucidate the molecular mechanism of reactions, location of these reaction sites on the amino acid sequence of the protein would be useful. We therefore first determined the mutation sites of various recA mutants newly isolated by us which are deficient in some or all of the recA functions and, based on the biochemical and biophysical properties of the mutant proteins as well as those of the modified RecA protein, we predicted the functional domains of the RecA protein. To learn how the reactions are done will require determination of the protein's three dimensional structure by X-ray crystallography. We are making effort to obtain a good quality of recA protein crystals suitable for detailed X-ray crystallographic analysis.