FLP SITE SPECIFIC RECOMBINASE

2微米圆质粒是在许多酵母菌株中发现的寄生元素。虽然该元件不编码任何正选择表位，但它能够保持稳定的拷贝数。该元件的维持需要位点特异性重组酶Flp的存在，Flp为诱导拷贝数扩增提供了一种开关机制。本应用程序解决了Flp功能的两个方面；重组反应的生化方向，以及调控Flp活性表达的机制。第一部分将解决Flp重组酶与单一底物结合时的顺式和反式切割问题，以及Holliday连接的跨水平和跨对角切割问题。这些问题将通过蛋白质靶向方法产生的裂解产物的体外分析来解决。本提案的第二部分在蛋白质-蛋白质相互作用水平上解决了Flp生化活性的调节。酵母- 2杂交系统的衍生物将用于分析Flp与其他2微米环蛋白的潜在关联，以及鉴定宿主细胞编码的调节蛋白。这些蛋白对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.