The proposed research should provide information about the molecular mechanisms and regulation of DNA repair, homologous recombination, and mutagenesis. These processes have numerous health consequences such as genetic disorders associated with defects in DNA repair (e.g., ataxia telangiectasia, Bloom's syndrome), drug resistance caused by recombination-dependent gene amplification, and the abundance of mutagenic agents that are carcinogenic. The specific focus of the proposal is the RecA protein of E. coli. This enzyme catalyzes the transfer of DNA strands between chromosomes in recombination and repair, induction of DNA repair genes, as well as other processes. The eucaryotic cognate of RecA has not yet been identified, hence E. coli uniquely allows the molecular details of these phenomena to be studied. Moreover, the powerful genetic techniques that can be applied in E. coli, such as gene fusion and recombinant DNA methodologies, make the RecA protein particularly susceptible to detailed analysis. The research will study the control of RecA synthesis, structure-function relationships within the RecA protein, and the consequences of genetically altering specific activities of this multifunctional enzyme on in vivo processes. Fusions between the recA and lacZ (encodes Beta-galactosidase) genes have allowed the requisite genetic procedures for this work to be developed. These methods have been used to isolate mutations affecting RecA synthesis and to identify functional domains of the RecA protein. The further use of these methods for a thorough genetic analysis of RecA is proposed. Additional mutations affecting synthesis will be isolated and analyzed to define transcriptional and translational control sites and determine the mechanisms and roles of these controls. Segments of the recA gene encoding functional domains of the protein will be identified by the phenotypes that they confer and subsequently mutationally altered. These mutations will be used to determine the physiological and biochemical functions of individual domains. In the long term, this analysis will provide a detailed picture of the synthesis and organization of the RecA protein, its enzymatic mechanisms, and its role in DNA metabolism.
