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 in eukaryotes using the yeast Saccharomyces cerevisiae as a model organism. Previous studies performed under the auspices of this project have led to the development and characterization of an in vitro system that uses extracts of mitotic cells to catalyze recombination events. Analysis of the in vitro recombination reaction has led to the purification of 4 proteins having properties consistent with their being involved in recombination. This analysis of recombination will be continued by following 7 lines of experimentation. 1) The biochemical properties of strand exchange protein 1 (SEP l) will be characterized using now available overproduced protein. 2) The properties conferred by sep1 mutations will be studied in greater detail to gain additional insights into the role SEP l plays in the cell. 3) SF1, an SEPI stimulatory factor that is also a strand exchange protein, will be overproduced and its biochemical properties determined in detail. 4) The effects of viable sfl insertion and deletion mutations on sporulation, recombination and repair will be determined. 5) The biochemical and genetic properties of intact heterotrimeric RPA, an SEP1 stimulatory factor, will be determined. 6) Additional proteins thought to be involved in catalyzing recombination will be purified and characterized. These include proteins such as endonucleases and exonucleases predicted by mechanistic studies of recombination and additional homologous pairing proteins, 3 additional already purified SEPI stimulatory factors and an already purified Holliday junction processing enzyme. And, 7) an already developed in vitro recombination system will be, used in conjunction with in vitro complementation and reconstitution approaches to identify additional recombination proteins. The ultimate goal of these studies will be to reconstitute recombination with purified proteins and defined DNA substrates and determine the mechanism(s) of these reactions.

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National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Dana-Farber Cancer Institute
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Page, A M; Davis, K; Molineux, C et al. (1998) Mutational analysis of exoribonuclease I from Saccharomyces cerevisiae. Nucleic Acids Res 26:3707-16
Johnson, A W; Kolodner, R D (1995) Synthetic lethality of sep1 (xrn1) ski2 and sep1 (xrn1) ski3 mutants of Saccharomyces cerevisiae is independent of killer virus and suggests a general role for these genes in translation control. Mol Cell Biol 15:2719-27
Tishkoff, D X; Rockmill, B; Roeder, G S et al. (1995) The sep1 mutant of Saccharomyces cerevisiae arrests in pachytene and is deficient in meiotic recombination. Genetics 139:495-509
Heyer, W D; Johnson, A W; Reinhart, U et al. (1995) Regulation and intracellular localization of Saccharomyces cerevisiae strand exchange protein 1 (Sep1/Xrn1/Kem1), a multifunctional exonuclease. Mol Cell Biol 15:2728-36
Johnon, A W; Kolodner, R D (1994) Characterization of the interaction of Saccharomyces cerevisiae strand exchange protein 1 with DNA. J Biol Chem 269:3673-81
Johnson, A W; Kolodner, R D (1994) The activity of the Saccharomyces cerevisiae strand exchange protein 1 intrinsic exonuclease during joint molecule formation. J Biol Chem 269:3664-72
Heyer, W D; Kolodner, R D (1993) Enzymology of homologous recombination in Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol 46:221-71
Alani, E; Thresher, R; Griffith, J D et al. (1992) Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein. J Mol Biol 227:54-71
Erdile, L F; Heyer, W D; Kolodner, R et al. (1991) Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication. J Biol Chem 266:12090-8
Tishkoff, D X; Johnson, A W; Kolodner, R D (1991) Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1. Mol Cell Biol 11:2593-608

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