Site-specific genetic recombination promoted by the FLP protein of the yeast 2 micron plasmid will be studied employing a defined, in vitro system. Purified FLP protein will be obtained from E. coli cells expressing the protein. The recombination site, characterized in earlier work, is available in a wide variety of altered forms, providing a number of experimental opportunities. Quantitative assays are in place. The work will focus on two primary objectives: a) An extensive kinetic analysis of this recombination event will be undertaken. Questions to be answered include but are not limited to: 1) How does FLP protein locate a recombination site, 2) How does FLP protein bring 2 such sites together, 3) Is FLP protein recycled after the reaction or does it only react once, 4) Can FLP protein carry out multiple reactions while bound to the same site? b) An auxiliary protein factor which is required for optimal FLP protein activity will be purified and an atempt will be made to identify it. Experiments will be carried out to determine its mode of action. Longer term goals include an increase in the production of purified FLP protein to facilitate physical and structural studies of this protein and its interaction with the recombination site. If the role of the auxiliary protein factor can be determined, an effort will be made to identify similar proteins in yeast. Finally, this recombination system will be developed as a reagent for use in certain types of DNA manipulations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037835-04
Application #
3293637
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-12-01
Project End
1991-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Huang, L C; Wood, E A; Cox, M M (1991) A bacterial model system for chromosomal targeting. Nucleic Acids Res 19:443-8
Qian, X H; Inman, R B; Cox, M M (1990) Protein-based asymmetry and protein-protein interactions in FLP recombinase-mediated site-specific recombination. J Biol Chem 265:21779-88
Meyer-Leon, L; Inman, R B; Cox, M M (1990) Characterization of Holliday structures in FLP protein-promoted site-specific recombination. Mol Cell Biol 10:235-42
Bruckner, R C; Cox, M M (1989) The histone-like H protein of Escherichia coli is ribosomal protein S3. Nucleic Acids Res 17:3145-61
Gates, C A; Cox, M M (1988) FLP recombinase is an enzyme. Proc Natl Acad Sci U S A 85:4628-32
Umlauf, S W; Cox, M M (1988) The functional significance of DNA sequence structure in a site-specific genetic recombination reaction. EMBO J 7:1845-52
Meyer-Leon, L; Huang, L C; Umlauf, S W et al. (1988) Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination. Mol Cell Biol 8:3784-96
Senecoff, J F; Rossmeissl, P J; Cox, M M (1988) DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site. J Mol Biol 201:405-21
Meyer-Leon, L; Gates, C A; Attwood, J M et al. (1987) Purification of the FLP site-specific recombinase by affinity chromatography and re-examination of basic properties of the system. Nucleic Acids Res 15:6469-88