Interactions of Escherichia coli UmuD with activated RecA analyzed by cross-linking UmuD monocysteine derivatives.
SOS mutagenesis in Escherichia coli requires the participation of a specialized system involving the activated form of UmuD (UmuD'), UmuC, RecA, and DNA polymerase III proteins. We have used a set of monocysteine derivatives of UmuD (M. H. Lee, T. Ohta, and G. C. Walker, J. Bacteriol. 176:4825-4837, 1994) and the cysteine-specific photoactive cross-linker p-azidoiodoacetanilide (AIA) to study not only the interactions of intact UmuD in the homodimer but also the interactions of UmuD with activated RecA. The reactivities of the individual UmuD monocysteine derivatives with AIA were similar to their reactivities with iodoacetate. The relative efficiencies of cross-linking of the AIA-modified monocysteine UmuD derivatives in the homodimer form are also consistent with our previous conclusions concerning the relative closeness of various UmuD residues to the dimer interface. With respect to the UmuD-RecA interface, the AIA-modified VC34 and SC81 monocysteine derivatives cross-linked most efficiently with RecA, indicating that positions 34 and 81 of UmuD are closer to the RecA interface than the other positions we tested. The AIA-modified SC57, SC67, and SC112 monocysteine derivatives cross-linked moderately efficiently with RecA. Neither C24, the wild-type UmuD that has a cysteine located at the Cys-24-Gly-25 cleavage site, nor SC60, the UmuD monocysteine derivative with a cysteine substitution at the position of the putative active-site residue, was able to cross-link with RecA, suggesting that RecA need not directly interact with residues involved in the cleavage reaction. SC19, located in the N-terminal fragment of UmuD that is cleaved, and LC44 also did not cross-link efficiently with RecA.
Lee
M H
MH
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Walker
G C
GC
D000083:Acetanilides / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003545:Cysteine; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D011938:Rec A Recombinases / Q000378:metabolism*
C097588:4-azidoiodoacetanilide; D000083:Acetanilides; D001386:Azides; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli; D003545:Cysteine