Probing the activation stages of the RecA protein by monoclonal IgGs during the pairing of homologous DNA molecules.
The biochemical properties of the RecA protein change in a stepwise manner with the binding of ATP and/or DNA during the course of the ATP-dependent formation of homologous joint molecules, which is assumed to be due to transition in the higher order structure. This transition was supposed to be detectable as changes in the affinity of the protein for monoclonal IgGs. In this study, we introduced an enzyme-linked immunosorbent assay, which enabled us to assay unbound IgG sensitively and quantitatively under the conditions for the RecA protein-mediated joint molecule formation. Through the use of this method, we studied the affinity of three anti-RecA protein monoclonal IgGs toward the RecA protein at the various stages of its activation during the formation of homologous joints. We found that the affinity of the RecA protein changed for each anti-RecA protein-IgG in a specific manner and the change in the cross-reactivity of the RecA protein correlated with its multiple activation stages. This result suggests that the RecA protein changes its higher order structure which is specific to each activation stage during the formation of homologous joint molecules.
Ikeda
M
M
Laboratory of Microbiology (Riken Institute, Institute of Physical and Chemical Research), Saitama, Japan.
Makino
O
O
Shibata
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D010584:Bacteriophage phi X 174 / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D007074:Immunoglobulin G; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011938:Rec A Recombinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011995:Recombination, Genetic
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D007074:Immunoglobulin G; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases