Genetic recombination between homologous DNA molecules in E. coli has been hypothesized to occur by three major nonbranched pathways, called the RecBC, RecE and RecF pathways. Recent data suggest that a network of interconnection branched pathways may be operative instead. In that light it is especially important to focus experimental work on the range of activities of individual recombination proteins. We are working on three such proteins and their genes, recA, recE and recF. With recA protein we plan to test a hypothesis about its three dimensional structure by detecting and studying recA mutations. One subset of mutations which inactivate the protein will be use to obtain additional mutations which restore activity. These will be mapped and checked against predictions of the tertiary structure hypothesis. Other subsets of recA mutations will be used for other purposes. For example the mutant proteins of a set of mutations which partially reverse the effects of recF mutations will be characterized to learn how they differ from wild type recA protein. The different properties are expected to be those of the missing recF protein. To check this, recF protein will be purified and characterized. Mutations affecting recF will also be sought to study the functions of the protein. We will complete the nucleotide sequence of the recE gene, its sbcA regulatory-gene and a set of genes coordinately regulated with recE. We will try to discover a predicted DNA strand renaturase activity in sbcA mutants and the gene encoding this activity if it exists. Finally we will investigate the application of modern methods to determine the kinetics of plasmid recombination. By looking early in the onset of recombination in various rec mutants we hope to detect and identify intermediates.
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