The purification and biochemical characterization of constitutive coprotease mutant RecA proteins as a tool for investigating structure -function relationships

The Escherichia coli RecA protein is essential for both homologous recombination and induction of the SOS response. The study of mutant RecA proteins is valuable in the understanding of the structure-function relationship of the RecA protein. It has been established that a parallel exists between the phenotypic behavior of recA mutant strains and the biochemical properties of the mutant proteins.;I describe here the purification and biochemical characterization of two mutant RecA proteins and discuss the implications of my findings on the structure-function relationship. The in vitro activity of the RecA P67W protein was examined to explain the constitutive induction of the SOS response found in vivo. This protein is characterized by an increase in ATP hydrolysis activity, an enhanced ability to invade ssDNA secondary structure, an increased formation of joint molecules in DNA strand exchange, and an enhanced ability to displace SSB protein, similar to RecA441 and RecA730 proteins. This is the result of an increased rate of association with ssDNA which allows RecA P67W protein to displace SSB protein from naturally occurring ssDNA and, hence, become activated for repressor cleavage in the absence of DNA damaging agents.;The second mutant RecA protein I present is the RecA N99 protein, which was characterized in vitro to explore the basis for its diminished recombination and constitutive coprotease phenotype. The ATP hydrolysis activity is attenuated and the association with ssDNA is slower. Binding of a second molecule of DNA to the nucleoprotein filament was not detected using DNA binding assays; however, joint molecules were formed in a DNA strand exchange assay, indicating binding in the secondary DNA binding site. These findings suggest an alteration in both the primary and secondary DNA binding sites which result in the decreased ability to perform recombinational repair. The in vitro LexA repressor cleavage activity of RecA N99 protein is reduced to approximately 60% of the wild type RecA protein level. The constitutive coprotease activity may be a result of the recombination deficiency, a consequence of the buildup of unrepaired DNA, which becomes available as a substrate for induction of the SOS response.