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 deletions 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 reaction. 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 existence of extensive interactions, possibly involving multiple 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.

RecN Is a Cohesin-like Protein That Stimulates Intermolecular DNA Interactions in Vitro

“…The concentration of the RecN protein (molecular mass, 59,798 Da) was determined from the absorbance at 280 nm using the calculated extinction coefficient 29,160 M Ϫ1 cm Ϫ1 . ATPase Assay-A coupled spectrophotometric enzyme assay was used to measure the ATPase activity of the RecN protein as described (48). The regeneration of ATP from phosphoenolpyruvate and ADP (produced by hydrolysis) was coupled to the oxidation of NADH through the addition of pyruvate kinase and lactate dehydrogenase.…”

SSB Antagonizes RecX-RecA Interaction

“…Enzymes and Reagents-The E. coli WT RecA protein was overexpressed and purified as described previously (40). The concentration of the purified protein was determined from the absorbance at 280 nm using the extinction coefficient 2.23 ϫ 10 4 M Ϫ1 cm Ϫ1 (60).…”

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

“…Enzymes and Biochemicals-The wild-type E. coli RecA, RecA⌬C6, RecA⌬C13, and RecA⌬C17 proteins were purified as described (23). A plasmid containing the recA E343K mutant (pEAW166) was constructed using PCR site-directed mutagenesis.…”

“…A plasmid containing the recA E343K mutant (pEAW166) was constructed using PCR site-directed mutagenesis. The RecA E343K point mutant protein was expressed and purified as described for the wildtype RecA protein (23). The concentrations of the purified RecA proteins were determined from the absorbance at 280 nm using the extinction coefficient of 2.23 ϫ 10 4 M Ϫ1 cm Ϫ1 (24).…”