A POSSIBLE ROLE OF THE C-TERMINAL DOMAIN OF THE RECA PROTEIN - A GATEWAY MODEL FOR DOUBLE-STRANDED DNA-BINDING

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270-328 (from the N terminus). Our model building pointed out the possibility that this do main is a part of ''gateway'' through which double-stranded DNA finds a path for direct contact with single-stranded DNA within a presynapti c RecA filament in the search for homology. To test this possible func tion of the domain, we made mutant RecA proteins by site-directed sing le (or double, in one case) replacement of 2 conserved basic amino aci d residues and 5 among 9 nonconserved basic amino acid residues in the domain. Replacement of either of the 2 conserved amino acid residues caused deficiencies in repair of W-damaged DNA, an in vivo function of RecA protein, whereas the replacement of most (except one) of the tes ted nonconserved ones gave little or no effect. Purified mutant RecA p roteins showed no (or only slight) deficiencies in the formation of pr esynaptic filaments as assessed by various assays. However, presynapti c filaments of both proteins that had replacement of a conserved amino acid residue had significant defects in binding to and pairing with d uplex DNA (secondary binding). These results are consistent with our m odel that the conserved amino acid residues in the C-terminal domain h ave a direct role in double-stranded DNA binding and that they constit ute a part of a gateway for homologous recognition.