INVESTIGATION OF THE SECONDARY DNA-BINDING SITE OF THE BACTERIAL RECOMBINASE RECA

The L2 loop is a DNA-binding site of RecA protein, a recombinase from Eschericha coil. Two DNA-binding sites have been functionally defined in this protein. To determine whether the L2 loop of RecA protein is p art of the primary or secondary binding site, we have constructed prot eins with site-specific mutations in the loop and investigated their b iological, biochemical, and DNA binding properties. The mutation E207Q inhibits DNA repair and homologous recombination in vivo and prevents DNA strand exchange in vitro (Larminat, F., Cazaux, C., Germanier, M. , and Defais, M. (1992) J. Bacteriol. 174, 6264-6269; Cazaux, C., Larm inat, F., Villani, G., Johnson, N. P., Schnarr, M., and Defais, M. (19 94) J. Biol. Chem. 269, 8246-8254). We have found that mutant protein RecA(E207Q) lacked one of the two single stranded DNA-binding sites of wild type RecA. The remaining site was functional, and biochemical ac tivities of the mutant protein were the same as wild type RecA with ss DNA in the primary binding site. The second mutation, E207K, reduced b ut did not eliminate DNA repair, SOS induction, and homologous recombi nation in vivo. In the presence of ATP, mutant protein RecA(E207K) cat alyzed DNA strand exchange in vitro at a slower rate than wild type pr otein, and ssDNA binding at site I was competitively inhibited. These results show that the L2 loop is or is part of the functional secondar y DNA-binding site of RecA protein.