This is a Shannon award providing partial support for the research projects that fall short of the assigned Institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. DESCRIPTION (adapted from the investigator's abstract): Genetic recombination is the process which moves DNA from one genomic location to another. After decades of genetic analysis, particularly in fungi, much is known about the DNA intermediates during recombination, By comparison, much less is known about the enzymes that generate these intermediates. The purpose of this proposal is to redress this imbalance by analyzing the enzymology of genetic recombination. The model system will be mating type switching in yeast. This was chosen for several reasons. First, mating type switching is a mitotic event and can therefore be studied in mitotic extracts. Biochemical analysis of meiotic recombination requires meiotic extracts, which are much more difficult to obtain in large amounts. Second, mating type switching occurs at an extremely high rate, making it possible to monitor progression by Southern blotting protocols. Third, HO endonuclease, the enzyme that initiates the reaction by forming a double strand break in DNA has been purified and partially characterized. Fourth, genetic analysis of mating type switching is highly developed. Finally, mating type switching depends on many of the same gene products as meiotic recombination. An understanding of the biochemical function of these gene products during the simpler mating type reaction will therefore indicate possible roles for these proteins in meiotic recombination. The experiments will focus on the initiation event of mating type switching -- the formation of a double strand break in DNA. The first part will analyze HO endonuclease in greater detail using biochemical and genetic methodologies. The second part will examine YZBP, a new protein isolated in the investigator's laboratory which has a binding site that overlaps with the recognition sequence for HO endonuclease. The function of YZBP will be examined both in vivo and in vitro using genetic and biochemical approaches. A detailed analysis of these two proteins will improve our understanding of the early steps of recombination, as well as the mechanisms used by cells to regulate the process. It should also provide valuable substrates to analyze later steps in the recombination reaction.
