Genetic recombination is an important aspect of nucleic acid metabolism. Besides bringing about genetic exchanges, recombination appears to be required for the accurate segregation of chromosomes during meiosis and plays an important role in the repair of various classes of DNA damage. Aberrant recombination events are associated with genetic defects and different types of cancer. Tumor promoters like TPA stimulate recombination and cell lines derived from individuals with Blooms syndrome and other diseases like ataxia telangiectasia appear to have elevated levels of recombination. Understanding the biochemical mechanism(s) of recombination is central to our knowledge of these diseases. While some aspects of the mechanism(s) of recombination are becoming clear, the overall enzymatic mechanism(s) of recombination is poorly understood. The long term goal of this proposal is to determine the enzymatic mechanisms of recombination using Escherichia coli as a model organism. Previous studies have used plasmid DNAs as substrates for studying homologous recombination. These studies demonstrated the plasmid recombination can be promoted by both the RecF recombination pathway and a recA-independent pathway called the RecE pathway, and have determined both the substrate specificity and the gene products required. To understand the biochemical mechanisms of these two recombination pathways, the two proteins encoded by the recE gene and the RecF, RecJ, RecN, RecO, RecQ, RecR, RuvA and RuvB proteins have been overproduced and, in most cases, purified. This analysis of recombination will be continued by following 5 lines of experimentation. 1) Additional genetic studies will continue to define the gene products required for recombination events promoted by the RecE and RecF recombination pathways. 2) A new system for analyzing the mechanism of recombination will be used to identify the DNA intermediates involved in the RecE and RecF pathways and to understand the role that individual recombination gene products play. 3) The biochemical and genetic properties of a protein that resembles the lambda beta protein and a second recombination protein encoded by the recE region, exoVIII, will be determined to help understand their role in recombination. 4) The proteins required for the RecF pathway encoded by the recF, recJ, recN, recO, recQ, recR, ruvA and ruvB genes have been overproduced and in most cases purified. The biochemical properties of these proteins will be determined and a major effort will be made to couple their actio with RecA and Ssb. And, 5) a major effort will be made to develop in vitro systems that use cell-free extracts to promote RecE and RecF pathway recombination. In vitro complementation and reconstitution approaches will be used to purify additional proteins required to reconstitute these reactions to permit a detailed analysis of recombination. The ultimate goal of these studies will be to reconstitute recombination with purified proteins and determine the mechanism(s) of these reactions.
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