9728557 Malone Meiosis is a required part of the life cycle for most eucaryotes; it is necessary for the production of haploid gametes. It has evolutionary importance as well, since much of the variation upon which natural selection acts is created during meiosis. There are a number of unique steps in meiosis, one of which is high levels of genetic recombination. Recombination is also required for proper chromosome pairing and for a proper first meiotic chromosome segregation. This research focuses on a region (a "hotspot") around the HIS2 gene in S. cerevisiae (baker's yeast) where recombination occurs frequently in meiosis. Correlated with meiotic recombination at HIS2 are high levels of two meiosis-specific double strand DNA breaks (DSBs). The distribution and properties of such breaks in meiosis, both at specific hotspots and along whole yeast chromosomes, is consistent with their role as intermediates in meiotic recombination. This project has two aims: 1 To finish the analysis of the cis-acting sequences required, and the breaks that occur, at HIS2, and 2 To define and study the trans-factors responsible for the HIS2 hotspot. The questions asked about the HIS2 hotspot in part of the research include: 1 How much DNA is sufficient for a hotspot to be recognized wherever it is located? 2 Is the transcriptional interference hypothesis correct at HIS2? 3 Is there a clear site (not sequence) specificity for DSB sites? 3 Is there competition between natural break sites, and does it occur in cis and/or in trans? 4 How many actual breaks occur in the region of the DSB? 5 Are the ends of the DSBs at hotspots flush or do they have 5' overlaps? Data from other hotspots gives two different answers. 6 Are all break sites (even those in coding regions) located in "open" chromatin regions? The second part of this research is aimed at finding factors which affect the HIS2 hotspot, but not recombination generally i.e., which create (or antagonize) the recombinogenic environ ment at HIS2 . Such factors might include at least three types of gene products: 1 Factors required for the long range chromatin structure of the region, 2 Factors required for short range chromatin structure at the DSB sites, and 3 Factors specific to the HIS2 locus not involved in chromatin structure. Any of these three types might affect other hotspots as well, and experiments will be conducted to ask that question.
