MOLECULAR MECHANISMS OF S POMBE MEIOTIC RECOMBINATION

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The long-term objective of the proposed research is to determine the basic molecular mechanism of meiotic homologous recombination in the fission yeast Schizosaccharomyces pombe. The specific aims are to 1) isolate and characterize mutants of S. Pombe defective in late steps of meiotic recombination such as resolution of Holliday structures formed during meiosis between homologous chromosomes and 2) determine, using molecular assays, S. Pombe meiotic recombination intermediates and products. Both genetic and biochemical approaches are used to achieve these objectives. In the genetic approach, a screen is proposed which will identify S. Pombe mutants defective in late steps of meiotic recombination. In this screen, a mutant involved in a late step of recombination and produces inviable spores is rescued by an additional early recombination mutant phenotype which prevents exchange from starting. In the biochemical approaches, molecular assays are used for detecting and analyzing S. pombe meiotic recombination intermediates (e.g., joint molecules, nicks, and double strand breaks) and mature products. These approaches use a two- dimensional DNA separation assay in combination with psoralen crosslinking of DNA, a primer extension assay for determining intermediates containing Holliday structures, and the mutants isolated from the genetic approach mentioned earlier. In eukaryotes, genetic recombination plays central roles in a) generating diversity at both the cellular and organismal levels, b) repair of damaged chromosomes, and c) faithful segregation of homologous chromosomes during meiosis. Recombinational aberrations generate DNA mutations (e.g., substitutions and deletions), and chromosome rearrangements (e.g., translocations) and loss (e.g., aneuploidy). Such aberrancies are often clinically manifested as cancer and birth defects in humans. S. pombe is used here as a model eukaryote to further our understanding of the molecular mechanisms of meiotic recombination. The knowledge gained here will give use more understanding into the causes of several diseases of genetic origin in humans (e.g., cancer) and possibly lead to insight into ways for preventing them in the future.