The long-term objective of work in this laboratory is to understand the mechanisms of cellular genetic recombination events. This proposal addresses the mechanism of FLP protein-promoted site-specific recombination, a reaction that occurs in the 2 micron plasmid of yeast. This was the first eukaryotic site-specific recombination event to be examined in vitro, and it provides a relatively simple system in which to study the chemistry of this reaction. Mechanistic studies will be carried out both in vitro and in vivo. The in vitro work, which represents about 75% of the work proposed, is designed to address a variety of unanswered questions concerning the reaction pathway. Additional protein chemistry will be done to define protein/DNA stoichiometries in the reaction and an attempt will be made to determine the structure of the protein. Kinetic studies will be used to determine if FLP protein can promote multiple reactions before dissociating. A closer look at protein-DNA interactions will be included, along with an effort to define DNA-DNA interactions during a key step in the reaction (the isomerization of the Holliday structure). The in vivo work will define experimental parameters that affect the use of the FLP system to promote planned DNA rearrangements in chromosomes. Most of the work will focus on chromosomal targeting of foreign DNA, and the initial efforts will be carried out with an FLP-mediated targeting system already developed for E. coli. The capacity to add or delete DNA at will in a cell, or in an organism during development, has much potential for improving our understanding of gene function, and the planned work should facilitate the use of FLP for a wide variety of studies. The health-related aspects of this work arise, first, from the importance of this or similar types of recombination in gene regulation, the generation of antibody diversity, recombination in Herpesvirus DNA, and other processes. The work also may lead to the development of improved techniques for manipulating cellular genomes, or gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037835-07
Application #
3293639
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-12-01
Project End
1995-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
7
Fiscal Year
1993
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