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 coli. Two DNA-binding sites have been functionally defined in this protein. To determine whether the L2 loop of RecA protein is part of the primary or secondary binding site, we have constructed proteins with site-specific mutations in the loop and investigated their biological, 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., Larminat, F., Villani, G., Johnson, N. P., Schnarr, M., and Defais, M. (1994) J. Biol. Chem. 269, 8246-8254). We have found that mutant protein RecAE207Q lacked one of the two single stranded DNA-binding sites of wild type RecA. The remaining site was functional, and biochemical activities of the mutant protein were the same as wild type RecA with ssDNA in the primary binding site. The second mutation, E207K, reduced but did not eliminate DNA repair, SOS induction, and homologous recombination in vivo. In the presence of ATP, mutant protein RecAE207K catalyzed DNA strand exchange in vitro at a slower rate than wild type protein, and ssDNA binding at site I was competitively inhibited. These results show that the L2 loop is or is part of the functional secondary DNA-binding site of RecA protein.
Cazaux
C
C
Institut de Pharmacologie et de Biologie Structurale du CNRS, 205, route de Narbonne, 31077 Toulouse Cedex, France.
Blanchet
J S
JS
Dupuis
D
D
Villani
G
G
Defais
M
M
Johnson
N P
NP
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011938:Rec A Recombinases