The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. Understanding the function of the RecA protein will reveal basic mechanisms which are at the foundation of general genetic recombination. Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer. It therefore assumes great clinical significance to understand the various mechanisms available to cells for the restoration of the integrity of their genetic material. Postreplication repair in prokaryotes corresponds to the filling of daughter strand gaps created by the arrest of replication at or near a lesion. Postreplication repair in E. coli is intimately associated with recombination and is dependent upon the RecA protein. Thus, studies of RecA polymers can be expected to help elucidate biological processes which range from meiosis to neoplastic transformation. Structural studies of RecA can make a large contribution towards such an understanding of function. An atomic model for RecA, from x-ray crystallography, has recently been published. The RecA protein, in the absence of DNA and ATP, forms a helix in the crystal that is similar to the active filament, formed by RecA on DNA with ATP. A key question in developing a molecular model for RecA function is understanding the conformational changes between the subunit in the crystal and in the active filament. Electron microscopy and image analysis can help provide this information. Specifically, studies will be undertaken to visualize the conformational changes between the crystal and the active filament, to look at the effect of mutations on low-resolution structure, and to visualize the interaction between RecA and its own repressor. These studies will provide a structural framework in which basic principles of general genetic recombination can be understood. The methods that are being used in this project can be readily applied to other systems, and two other projects, visualization of DNA topology in ice and DNA packaging in a bacteriophage, will be continued.
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