The goal of this proposal is to determine the enzymatic mechanisms of genetic recombination and repair of mismatched nucleotides in the yeast Saccharomyces cerevisiae. The basic approach that is being followed is to develop in vitro systems and biochemical assays that measure genetic recombination and mismatch repair in order to identify the biochemical intermediates and purify the proteins that are involved in genetic recombination and mismatch repair. Plasmid DNA and M13 phage DNA substrates that allow biochemical and genetic detection of homology dependent recombination events and the repair of mismatched nucleotides have been constructed and used to detect recombination and mismatch repair catalyzed by cell-free extracts of mitotic cells. The intermediates and products that are formed in these reactions will be characterized in detail to provide insight into the mechanisms of these reactions. Recombination substrates treated with mutagens, carcinogens, Gamma-irradiation and UV light will be constructed to access the effect of DNA damage on recombination in vitro. The effect of chromatin structure and the presence of recombination hotspots on recombination in vitro will be determined and a comprehensive study of the specificity of mismatch repair in vitro will be carried out. Cell-free recombination systems using extracts prepared form either meiotic cells or Gamma-irradiation or mutagen-induced cells will be developed and characterized. The proteins required for recombination and mismatch repair will be purified using a combination of in vitro complementation and reconstitution assays and characterized in detail. Studies on the purification and characterization of a S. cerevisiae Holliday Junction specific endonuclease will continue. The genes encoding proteins required for recombination and mismatch repair will be cloned in order to overproduce their gene products and to facilitate genetic analysis of the proteins to determine if they are required for recombination and mismatch repair in vivo. The ultimate goal of these studies will be to reconstitute recombination and mismatch repair reactions with purified proteins and determine the enzymatic mechanisms of these reactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029383-08
Application #
3276982
Study Section
Genetics Study Section (GEN)
Project Start
1983-07-01
Project End
1991-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
8
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
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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