Functional organization of the Escherichia coli RecA protein: Alteration of the nucleoside triphosphate catalytic domain, indentification of single-stranded DNA binding site(s), and cleavage of the LexA repressor

Critical to genetic recombination and inducible responses to DNA damage in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of LexA repressor, respectively. Since both activities require ssDNA and ATP, they display many parallels, including stimulation by SSB protein. However, unexpectedly, SSB protein inhibits LexA proteolysis and ATP hydrolysis (in the presence of repressor) at concentrations of RecA protein which are substoichiometric to the ssDNA, arguing that LexA repressor alters the competition between RecA and SSB proteins for limited ssDNA binding sites. Inhibition of LexA repressor cleavage by dsDNA or with an excess of ssDNA suggests that interaction of the RecA protein-ATP-ssDNA complex with either LexA repressor or a secondary DNA molecule is mutually exclusive. Thus, genetic recombination and SOS induction are intrinsically competitive processes.; Hydrolysis of nucleoside triphosphates, such as ATP or GTP, plays a central role in a variety of biochemical processes but, in most cases, the specific mechanism of energy transduction is unclear. DNA strand exchange promoted by RecA protein is normally associated with ATP hydrolysis. Conservative substitution of arginine for the invariant lysine-72 of RecA protein reduces NTP hydrolysis by {dollar}sim{dollar}600-850-fold. Despite this dramatic attenuation in hydrolysis activity, RecA K72R protein promotes efficient homologous pairing and extensive exchange of DNA strands. These results identify a component of the catalytic domain for NTP hydrolysis and substantiate the idea that RecA protein-promoted pairing and exchange of DNA strands mechanistically requires the allosteric transition induced by NTP cofactor binding, but not the energy derived from NTP hydrolysis.; Specific photochemical crosslinking identifies three peptides of RecA protein (amino acids 61-72, 178-183, and 233-243) which are proximal to and may mediate binding, and therefore subsequent pairing, of DNA. Their structural location and surface accessibility, along with the behavior of various recA mutants, support assignment of these regions to DNA binding site(s) of RecA protein. Functional overlap of amino acids 61-72 with an element of the ATP binding site suggests a structural mechanism by which nucleotide cofactors allosterically modulate the RecA nucleoprotein filament.