RECOMBINATION IN VITRO: ENZYMOLOGY AND INTERMEDIATES

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The enzymology of homologous pairing and strand exchange promoted by E. coli recA protein's unique. Whereas replication and transcription involve multi-enzyme complexes built from large sets of different polypeptide chains, recA protein polymerizes on single-stranded DNA to treat a multimolecular and helical enzymatic machine that is capable of promoting homologous pairing and strand exchange. Only one other protein, E. coli single-stranded DNA binding protein (SSB) is known to participate directly with recA protein in pairing and strand exchange, but other enzymes must create the broken DNA substrates on which recA protein can act, and must resolve the intermediates created by recA protein. The long term objective of the research proposed here is to define enzymatically the chain of events that constitute the major pathway of homologous recombination in E. coli. One means to that end, and an intermediate goal is the complete reconstitution of homologous recombination in vitro from purified enzymes. Our short range goals are: 1) to set up and develop the assay based on the packaging of lambda DNA in order to detect sensitively recombination of DNA molecules in crude extracts of E. coli, 2) through the use of particular constructs of phage lambda for crosses in known mutants of E. coli, to elucidate as far as possible the set of enzymes that are required in vivo for homologous recombination controlled by the principal pathway of recombination, the recABCD pathway, 3) to use the packaging assay (item I above and the in vivo observations (item 2 above) to develop an in vitro system starting from crude extracts. as a means to purify and identify a minimal set of enzymes required for homologous recombination, 4) to study mutational changes in the entire set of known recombine activities of recA protein purified from new recA mutants in order to analyze the mechanism of homologous pairing and strand exchange, and 5) to analyze further the topological requirements, the changes in DNA conformation, and the intermediates that are involved in the homologous pairing of naked duplex DNA with the helical recA nucleoprotein filament. Homologous recombination, one of the keystones of genetics, is a subject of fundamental interest and importance. The use of homologous recombination to "target" genes to specific chromosomal sites in mammalian cells may significantly expand the genetic tools available to study mammalian cells and may eventually provide one of the means necessary to accomplish gene therapy.