C-terminal Deletions of the Escherichia coli RecA Protein

Abstract

A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal dele-tions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reac-tion. However, at the same time, the deletion of 13–17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the exist-ence of extensive interactions, possibly involving multi-ple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK a of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.