RecA-mediated cleavage reaction of Lambda repressor and DNA strand exchange require an active extended filament conformation but not ATP hydrolysis

Abstract

DNA pairing and strand exchange activities are essential for genetic recombination. When DNA is damaged, RecA proteins bind to DNA in the presence of ATP and catalyze the specific proteolytic cleavage of Lambda repressor. The cleavage reaction induces and regulates the expression of DNA repair genes. In this work, it has been examined by introducing sites directed mutagenesis (in the ATP catalytic domain or in the DNA binding loop of RecA), the ability of RecA protein to hydrolyze ATP or to
cleave Lambda repressor either in the presence of DNA or in the presence of high salt concentration, and the ability of RecA to promote DNA strand exchange. It was observed that mutant E96D does not hydrolyze ATP at all, but fulfills RecA functions such as cleavage of Lambda repressor and strand exchange in the presence of DNA. However mutant E158K hydrolyzes ATP as well in the presence of high salt concentration as in the presence of DNA, but does not fulfill RecA functions. These observations suggest that ATP hydrolysis is not required for the cleavage  of Lambda repressor and the genetic recombination, but is necessary for the release of RecA from DNA before DNA repair.