Epitopes and active sites of the RecA protein.
The RecA protein is indispensable for homologous genetic recombination in Escherichia coli. This protein alone promotes the ATP-dependent formation of homologous joint molecules and their processing in vitro. Through the use of a set of anti-RecA protein mouse monoclonal IgGs, we have been attempting to divide the whole process into elementary steps to determine the basic functions of the protein. In order to correlate the basic functions with the active sites on the recA polypeptide, we located the epitopes for the anti-RecA protein-IgGs on the recA polypeptide by means of immunoblotting experiments and an enzyme-linked immunosorbent assay involving isolated proteolytic polypeptides or synthetic ones derived from various regions of the recA polypeptide. The epitopes for anti-RecA protein-IgGs ARM321 and ARM414, both of which are shown to inhibit the DNA-dependent ATP hydrolysis and the formation of homologous joints by the RecA protein, were found to be located between Thr89 and Glu127 and between Glu233 and Lys256, respectively, on the RecA polypeptide. IgG ARM193 had been shown to interfere with the protein-protein interaction between two RecA protein molecules, and ARM191 had been suggested to inhibit the binding of double-stranded DNA to the RecA protein. The epitopes for ARM193 and ARM191 were found to be located in a approximately 90-amino acid region at the C terminus. These results suggest the locations of the active sites and a functional core on the RecA polypeptide.
Laboratory of Microbiology, Riken Institute of Physical and Chemical Research, Saitama, Japan.
M
M
Ikeda
O
O
Makino
T
T
Shibata
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D007074:Immunoglobulin G; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D011938:Rec A Recombinases / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D007074:Immunoglobulin G; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D010447:Peptide Hydrolases