The long-term objective of the research proposed here is to determine the mechanism of generalized genetic recombination in terms of the enzymes promoting the individual steps of this process and the structures of the DNA intermediates at each step. An additional objective is to determine the mechanisms by which recombination is regulated. We propose to develop a cell-free system for recombination derived from Escherichia coli. Our substrate for recombination will be DNA from a derivative of phage Lambda that may undergo intramolecular recombination between inverted repeats. Following incubation in an E. coli extract, this DNA will be converted by in vitro packaging into phage which can be assayed by selective plating for parental and recombinant types. Our preliminary investigations and those of others demonstrate that in infected cells this recombination-mediated inversion requires identified E. coli recombinational enzymes - RecA and RecBC - and is stimulated by Chi sites. We will determine the requirement for additional proteins, such as DNA ligase and single-strand binding protein (SSB), and for other processes, such as DNA replication. With the cell-free system, we will identify any additional required components and determine the structures of the DNA intermediates. We will continue our studies of the RecBC enzyme of E. coli to determine the mechanism by which it unwinds and rewinds DNA. We will determine RecBC's of DNA, and its rates of unwinding and rewinding in the presence of other proteins, such as RecA and SSB, known to be involved in recombination. Through a study of the molecular genetics of recBC we will investigate the regulation of expression of the recBC genes, develop a fine structure map of the recBC genes, continue work with Lambda recBC clones isolated in our preliminary work, and seek recBC mutants to evaluate the physiological roles of RecBC enzyme and the mechanisms that regulate its synthesis and activity.
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