FLP SITE SPECIFIC RECOMBINASE

2微米环质粒是许多酵母菌的寄生因子。虽然该元件不编码任何正选择表位，但它能够维持稳定的拷贝数。维持这一元件需要存在一种位点特异性重组酶FLP，它为诱导拷贝数扩增提供了一种切换机制。本申请涉及FLP功能的两个方面：重组反应的生化定位和FLP活性表达的调节机制。第一部分将讨论当FLP重组酶结合到单个底物上时，FLP重组酶的顺式和反式切割的问题，以及Holliday连接的横向和横向切割的问题。这些问题将通过对蛋白质靶向方法产生的切割产物的体外分析来解决。该建议的第二部分阐述了在蛋白质-蛋白质相互作用水平上对FLP生化活性的调节。酵母双杂交系统的一个衍生物将被用来分析FLP与其他2微米环状蛋白的潜在联系，以及识别由宿主细胞编码的调节蛋白。这些蛋白质对FLP重组活性的影响将在体外进行分析。

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.