The proposed experiments will exploit the unique biology of the mushroom Coprinus cinereus to understand the role of DNA repair genes in related processes of meiotic DNA metabolism. The repair of DNA damage and the proper segregation of homologs in meiosis are two fundamental activities of eukaryotes, and both are of paramount importance in human health. Defects in DNA damage processing lead to chromosomal rearrangements, mutation, and cancer. Defects in meiotic chromosome segregation lead to aneuploidy and are responsible for birth defects and spontaneous abortion. It is striking that these two important processes are genetically and mechanistically related. The study of DNA repair and meiosis in C. cinereus is exceptional, in that it combines the power of haploid fungal genetics and molecular biology with the ability to examine meiotic functions within the context of a multicellular organism in which meiosis is naturally synchronous. The C. cinereus rad3-9-11-12 epistasis group has been shown by this laboratory to be necessary for both meiosis and for the survival of gamma irradiation. During the proposed funding period, the following studies will be pursued: 1. Analysis of the function of the Rad9 protein and its homologs. Goals include the demonstration of functional conservation between Rad9 and its Saccharomyces cerevisiae ortholog, Scc2, an investigation of Rad9 protein location and interactions within the meiotic cell, and analysis of defects caused by the rad9-1 mutation. 2. Determination of the role of the Mre11 (Rad11) protein during meiosis in C. cinereus. This will include immunolocalization of the Mre11 protein in meiotic cells and the use of Mre11 phosphoesterase motif mutants to investigate the role of DNA double-strand breaks during meiosis in C. cinereus. 3. Investigation of double-strand break (DSB) repair in C. cinereus. The I-SceI endonuclease system will be used to create an inducible DSB at a defined locus, and the types of DSB repair employed by wild-type and rad mutant cells will be determined, with an initial focus on rad9-1 and rad11-1 (mre11-1). 4. Isolation and analysis of rad3 and rad12, the remaining two genes of the rad3-9-11-12 epistasis group. 5. Isolation and analysis of other genes whose functions are affected by genes of the rad3-9-11-12 group.
