Investigation of the Secondary DNA-binding Site of the Bacterial Recombinase RecA

摘要: The L2 loop is a DNA-binding site of RecA protein, recombinase from Eschericha coli. Two sites have been functionally defined in this protein. To determine whether the protein part primary or secondary binding site, we constructed proteins with site-specific mutations and investigated their biological, biochemical, DNA properties. mutation E207Q inhibits repair homologous recombination vivo prevents strand exchange vitro (Larminat, F., Cazaux, C., Germanier, M., Defais, M. (1992) J. Bacteriol. 174, 6264–6269; Larminat, Villani, G., Johnson, N. P., Schnarr, (1994) Biol. Chem. 269, 8246–8254). We found that mutant RecAE207Qlacked one two single stranded wild type RecA. remaining was functional, biochemical activities were same as ssDNA site. second mutation, E207K, reduced but did not eliminate repair, SOS induction, recombinationin vivo. In presence ATP, RecAE207K catalyzed at slower rate than I competitively inhibited. These results show functional