Site-specific genetic recombination promoted by the FLP protein of the yeast 2 micron plasmid will be studied employing a defined, in vitro system. Purified FLP protein will be obtained from E. coli cells expressing the protein. The recombination site, characterized in earlier work, is available in a wide variety of altered forms, providing a number of experimental opportunities. Quantitative assays are in place. The work will focus on two primary objectives: a) An extensive kinetic analysis of this recombination event will be undertaken. Questions to be answered include but are not limited to: 1) How does FLP protein locate a recombination site, 2) How does FLP protein bring 2 such sites together, 3) Is FLP protein recycled after the reaction or does it only react once, 4) Can FLP protein carry out multiple reactions while bound to the same site? b) An auxiliary protein factor which is required for optimal FLP protein activity will be purified and an atempt will be made to identify it. Experiments will be carried out to determine its mode of action. Longer term goals include an increase in the production of purified FLP protein to facilitate physical and structural studies of this protein and its interaction with the recombination site. If the role of the auxiliary protein factor can be determined, an effort will be made to identify similar proteins in yeast. Finally, this recombination system will be developed as a reagent for use in certain types of DNA manipulations.
