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.
