Inactivation of the recA protein by mutation of histidine 97 or lysine 248 at the subunit interface.

We have used site-directed mutagenesis to prepare two new mutant recA proteins, one in which histidine 97 has been replaced by alanine, and another in which lysine 248 has been replaced by alanine. Although these mutant proteins were originally designed from different considerations, they turned out to have remarkably similar properties. Both the [H97A]recA protein and the [K248A]recA protein bind poorly to single-stranded DNA, have no single-stranded DNA-dependent ATP hydrolysis activity, and do not promote renaturation of complementary single-stranded DNA molecules or the ATP-dependent three-strand exchange reaction. Furthermore, both mutant proteins are defective in Mg(2+)-induced helical filament formation. To account for these results, we propose that the mutation of either histidine 97 or lysine 248 alters subunit interactions between recA monomers and that this leads to the loss of cooperative single-stranded DNA binding and DNA pairing activities. This proposal is consistent with the recently determined x-ray structure of the recA protein, which shows that although histidine 97 and lysine 248 are distant from one another in the monomer structure, these two residues are on the opposing complementary faces of the recA subunit and pack against each other at the interface between adjacent recA monomers in the helical filament (Story, R. M., Weber, I. T., and Steitz, T. A. (1992) Nature 355, 318-325).