Genetic recombination is an important DNA metabolic pathway. The recombination apparatus is involved in the repair of DNA damage, increases meiotic genetic fitness, is required for chromosome segregation and provides the enzymatic activities for gene targeting and gene therapy. The objective of this proposal is to detail the biochemistry and molecular biology of genetic recombination in human cells. Previous studies have identified five proteins that affect recombination in vitro: HPP-1, hRP-A, NHR-ligase, A1 and SF-1. Specific methods have been developed to purify HPP-1, hRP-A, and A1 to near homogeneity from a defined human tumor cell line. We will investigate other cellular sources to improve purification and reduce the costs associated with large-scale tissue culture. Initial experiments will complete the biochemical characterization of these purified components and continue the purification of NHR-ligase and SF-1. We propose to further study these recombination proteins by cloning and sequencing their genes, and ultimately overexpressing the gene products in a heterologous system to aid in their purification and characterization. A reagent source of recombination proteins will be developed and used to study their interaction with normal DNA and DNA containing specific radiation and chemically induced lesions. An important thrust of these studies will be to determine the biological significance of these recombination proteins in human cells. The ultimate goal of these studies is to identify other recombination associated components toward the long-term goal of reconstituting the complete human recombination reaction in vitro. An understanding of the human recombination process should eventually allow for specific intervention in the recombination-repair process so as to increase therapeutic efficacy following radiation treatment and potentiate the techniques required for gene therapy.
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