The protein encoded by the recA gene of E. coli, Eco RecAp, is of paramount importance in several processes of DNA metabolism: (1) general genetic recombination (2) repair of chemical and irradiation damage to DNA, (3) UV mutagenesis and (4) coordinating repair, replication, and cell division after DNA has been damaged. Analogues of recA with similar activity have been found in five bacterial species closely related to E. coli and nine distantly related species. Proteins with activities of EcoRecAp have now been isolated from fungal, insect, plant and mammalian cells. Of all these EcoRecAp has been most thoroughly studied genetically and enzymatically. Still, however, we have only limited insight into the structure of EcoRecAp and how that structure relates to enzymatic activity. The standard technique of x-ray crystallography has been applied to EcoRecAp but has so far been unsuccessful in revealing its structure because of the flexibility of the protein. Computer analysis has detected similarities of the central part of EcoRecAp with a homogeneous group of proteins, called parallel aB proteins. Half of this proposal consists of genetic tests of the three-dimensional model of this central part, which has been called the core. The tests consist in part of averaging predictions for RecAps from several species, a method used successfully to predict the three-dimensional structure of another parallel aB protein. The test also consist in part of examining the predicted results of altering specific amino acids. These tests will help refine the model, whether the predictions are confirmed or contradicted, by a process akin to successive approximations. The other half of the proposal concerns the structural and functional roles of the amino and carboxy terminal regions of EcoRecAp. These are the regions whose structure was not predicted in the model of the core. Two monoclonal antibodies will be studied for their effects on EcoRecAp. One, MAb4B4, binds to amino terminal amino acids; the other, MAb156, binds to carboxy terminal amino acids. Binding competition of the two antibodies may reveal how closely these regions are situated in the structure of EcoRecAp. Effects of each antibody on six in vitro activities of EcoRecAp may reveal how these regions participate in the functions of the protein.
