Homologous genetic recombination is a process common to most organisms and is involved in many aspects of DNA metabolism. In addition, the specific introduction of foreign DNA into eukaryotic chromosomes, a basic step in the developing area of gene therapy, is critically dependent on the cell's genetic recombination machinery. For these reasons and others, it is desirable to obtain a detailed understanding of this process. Our long-term goal is to reconstitute homologous recombination in vitro with highly purified proteins, using the bacteriophage T4 as a useful model system. This proposal describes experiments directed towards this goal. We will continue to study a novel four protein strand exchange """"""""machine"""""""" composed of the purified phage uvsX (a RecA-like recombinase), gene 32 (a helixdestabilizing protein), uvsY (an accessory factor) and dda (a DNA helicase) gene products. This is the most biologically relevant system available for the in vitro study of synapsis and branch migration. Indeed, analogues of the latter two factors have not yet been characterized in any other organisms and the T4 studies are providing a wealth of new information on the roles of recombination accessory factors. In addition, we will purify and study as yet uncharacterized proteins that genetic and biochemical studies have implicated as being involved in T4 homologous recombination. With these factors in hand, we hope to be able to reconstitute a complete recombination reaction between completely duplex DNAs in vitro. The interplay between homologous recombination and DNA replication and transcription will also be assessed in vitro. We have been able to reconstitute a replication-dependent homologous pairing reaction and will further develop this novel system. The ability of DNA-binding transcriptional regulatory factors to block protein-mediated branch migrations will be assessed. Finally, we will begin to map specific functions of the uvsX protein to specific domains through the biochemical study of fragments produced proteolytically or genetically. These experiments will initiate an effort to understand this multifunctional protein at a chemical and structural level.
