I propose to study the mechanism of genetic recombination in E. coli using plasmid DNAs as recombination substrates. There are two pathways of plasmid recombination in E. coli. One pathway is blocked by mutations in the recA, recF andtopoisomerase I genes and is partially blocked by recB recC mutations. Recombination by this pathway can be stimulated when the plasmid substrates contain either of two classes of cloned DNA segments: one of which only act in cis and the other of which acts in trans. The second pathway is induced in recB recC strains by the sbcA mutation and is independent of recA or recF function. Derivatives of plasmids containing mutations which modify restriction endonuclease cleavage sites and inactivate either tetracycline resistance or ampicillin resistance genes have been constructed. These plasmids will be used in in vivo experiments to measure the rate of recombination, to study the mechanism of recombination and to assess the effects of recombination-deficient mutations on recombination. New recombination-deficient mutations affecting plasmid recombination will be isolated using a new screening procedure and mapped. The effect of these mutations on recombination will be determined. The recombination genes will be cloned to identify, overproduce and purify their gene products. The mechanism by which DNA sequences can stimulate plasmid recombination in cis will be studied and the active nucleotide sequence of these recombination hotspots will be determined. The mechanism by which some cloned DNA segments can stimulate plasmid recombination in trans will also be studied. Assays using plasmids to detect intragenic recombination and the recombination interconversion of plasmids and their circular oligomeric forms in vitro have been developed. Previously unidentified proteins involved in the plasmid recombination pathways will be purified and characterized using in vitro complementation assays and in vitro reconstitution assays. Studies on the mechanism of action of exonuclease VIII, a protein required for the sbcA-induced pathway, will be continued. The recombination pathways will be reconstituted with purified proteins to study the mechanism of plasmid recombination. The interaction between the purified recombination proteins and recombination hotspots will be studied in detail.
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