C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects.
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.
Lusetti
Shelley L
SL
Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA.
Wood
Elizabeth A
EA
Fleming
Christopher D
CD
Modica
Michael J
MJ
Korth
Joshua
J
Abbott
Lily
L
Dwyer
David W
DW
Roca
Alberto I
AI
Inman
Ross B
RB
Cox
Michael M
MM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D010584:Bacteriophage phi X 174 / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D016685:Mitomycin / Q000494:pharmacology; D011938:Rec A Recombinases / Q000737:chemistry*
D004270:DNA, Circular; D029968:Escherichia coli Proteins; D016685:Mitomycin; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases