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 correct processing and 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 addition, the meiotic process is naturally highly synchronous in C. cinereus, and meiotic chromosomes are amenable to analysis by microscopic and molecular techniques. During the proposed funding period, the following experiments will be pursued: 1. A detailed analysis of rad9, a novel gene necessary for both gamma ray survival and meiosis which was cloned and characterized during the current funding period. This analysis will include an in-depth phenotypic characterization of the rad9-1 mutation, the isolation and characterization of multiple defined alleles of this locus, studies of the time and location of expression of the rad9 protein, and genetic assays for direct interaction between rad9 and other C. cinereus rad genes. 2. The isolation and characterization of other rad genes of C. cinereus, using the complementary approaches of cloning genes identified by mutant screens for rad mutants, and cloning homologs of rad genes identified in other organisms. Efforts will focus on the C. cinereus gene radl2, and on homologs of the recA gene of Escherichia coli and the RAD52 gene of Saccharomyces cerevisiae. 3. A detailed study of the phenotypes of those rad mutants that also have meiotic disfunction. The processes analyzed will include pre-meiotic DNA replication, meiotic chromosome alignment and synapsis, double-strand break repair, mitotic recombination within the tandemly repeated ribosomal RNA genes, and MIPPING (methylation induced pre-meiotically). 4. Genetic screens for extragenic suppressors of the meiotic defects of rad mutants, and genetic tests for direct interactions between pairs of rad gene products.
