RECOMBINATION IN VITRO--ENZYMOLOGY AND INTERMEDIATES

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E. Coli RecA protein forms a right-handed helical nucleoprotein filament on single-stranded DNA which catalyzes a search for homology, assimilates homologous duplex DNA, and effects the complete exchange of strands to produce a new heteroduplex recombinant. This protein appears to be the prototype of homologous recombination proteins that have recently been found to be universally distribute in nature, including Homo sapiens. The university of such proteins underscores the importance of understanding their molecular mechanisms, which may be important in disease processes that involve derangements of repair and recombination, and which will be of increasing interest in regard to gene targeting and gene therapy. The mechanisms of homologous pairing and strand exchange via the helical recombination filament are still not well understood, but intermediates have been identified and isolated that permit further enzymological and chemical probing. Recent studies have revealed that RecA protein can catalyze homologous recognition through the formation of non-Watson-Crick bonds. Experiments are proposed to probe the nature and role of these intermediates to test the hypothesis that homologous recognition is accomplished by a non-Watson-Crick interaction between a RecA coated single strand and duplex DNA. The rotation of DNA has been shown to be important in strand exchange. Experiments will test the hypothesis that rotation is accomplished by the coaxial rotation of DNA within the nucleoprotein filament. Experiments in vitro reveal the strand exchange phase of the recombination reaction only when a substrate is provided with an appropriate free end. A search will be made for enzymes that will cleave the initial synaptic complex made by RecA protein into one that can progress to the next stage of strand exchange. To this end, substrates and assays will be developed that can be used in crude extracts. Such methods may prove useful in the enzymolifocal approach to homologs of RecA protein in eukaryotes. The short term goals of the proposed research are to gain understanding of the mechanisms of homologous recognition and strand exchange and to discover how the intermediate that is generated by RecA protein is further processed. The long term goals are to understand the pathways of recombination mediated by RecA protein and its homologs, and to use that knowledge to facilitate gene targeting and gene therapy.