PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF ESCHERICHIA-COLI RECA PROTEINS MUTATED IN THE PUTATIVE DNA-BINDING SITE

Escherichia coli RecA protein plays a central role both in DNA repair and in recombination. We report biochemical properties of three new Re cA proteins mutated at positions 199 (RecA694), 207 (RecA659), and 211 (RecA611) in the putative DNA binding site. RecA694 had a wild-type p henotype, whereas RecA611 and RecA659 were deficient in promoting both the self-cleavage of LexA repressor and the DNA-strand exchange react ion. In order to determine the origin of this inhibition, we examined the capacity of wild-type and mutant proteins to bind to single-strand ed DNA (with and without single- stranded binding protein, SSB), doubl e-stranded DNA, and ATP. DNA strand exchange defects were correlated w ith the inability of mutant proteins to displace SSB from DNA. For the recA659 mutation this inhibition was reversed by equimolar wild-type protein. In contrast, mixtures of either wild-type/RecA659 or wild-typ e/ RecA611 proteins remained deficient in LexA cleavage, suggesting th at the dominant negative phenotype of the mutant proteins may be a con sequence of the formation heterologous RecA complexes. Various mutatio ns in the putative DNA binding site of RecA protein altered ATP bindin g, ATPase activity, displacement of SSB from single-stranded DNA, and protein-protein interactions. These results are consistent with the hy pothesis that DNA binding to this site of RecA relays allosteric effec ts to several functional domains throughout the protein.