The 2 micron circle plasmid is a parasitic element found in many strains of Saccharomyces. Although this element does not encode any positive selection epitopes, it is able to maintain a steady-state copy number. Maintenance of this element requires the presence of a site-specific recombinase, Flp, which provides a switching mechanism for induction of copy-number amplification. This application addresses two aspects of Flp function; the biochemical orientation of the recombination reaction, and the mechanism by which the expression of Flp activity is regulated. Part one will address the question of cis verses trans cleavage of the Flp recombinase when bound to single substrates, and trans-horizontal verses trans-diagonal cleavage of Holliday junctions. These questions will be approached using in vitro analysis of cleavage products resulting from a protein targeting method. Part two of this proposal addresses regulation of Flp biochemical activity at the level of protein-protein interactions. A derivative of the yeast-two hybrid system will be used to analyze the potential association of Flp with other 2 micron circle proteins, as well as to identify regulatory proteins encoded by the host cell. Effects of these proteins of Flp recombination activity will then be analyzed in vitro.
