RECOMBINATION IN VITRO: ENZYMOLOGY AND INTERMEDIATES

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E. coli recA protein in vitro promotes homologous pairing of single-stranded or partially single-stranded DNA in three sequential steps: 1) presynaptic polymerization of recA protein on single-stranded DNA, 2) synapsis, the conjunction and homologous alignment of DNA molecules, and 3) strand exchange. Three nucleoprotein structures have been identified as putative intermediates: 1) presynaptic complexes, single-stranded DNA with recA protein polymerized on it, 2) conjoined molecules, double-stranded DNA bound to presynaptic complexes without homologous pairing, and 3) synaptic or nascent heteroduplex structures, novel three-stranded intermediates in which the incoming strand is not topologically interwound with its complement but is nonetheless base-paired via a nascent heteroduplex joint. By enzymological and biochemical methods, we will explore the structures of these intermediates, aiming specifically at defining the points of contact of the strands of DNA and recA protein. We have studied the interactions of reCA protein with the following enzymes from E. coli: single-strand binding protein, T4 gene 32 protein, topoisomerase I, DNA ligase, recBC DNase, exonuclease I and Lambda exonuclease. By a series of model systems involving various DNA substrates and various combinations of enzymens we propose to develop recombination assays that are suitable for use with crude extracts or fractions thereof in order to reconstitute recombination in vitro and thereby to enable the identification of all of the enzymens of the major pathway of homologous recombination in E. coli. For this purpose, recA protein provides an instrumental reagent, since it both brings DNA molecules together and puts them in homologous alignment. On the one hand no single pathway of recombination has yet been elucidated; on the other hand, research made possible in recent years by recombinant DNA methodology has revealed the importance of recombination not only in inheritance but also in biological regulation and development.