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 C. cinereus rad3-9-11-12 epistasis group most likely comprises C. cinereus orthologs of the Mre11/Rad50 double-strand break repair complex, plus an adherin protein (Rad9) necessary for meiotic sister chromatid cohesion and homolog pairing. During the proposed funding period, the following studies will be pursued: 1. Analysis of meiotic functions and interactions of the Mre11/Rad50 complex. We will analyze the effects of defined changes in the Rad50 protein on meiotic DSB formation, meiotic and mitotic interactions with Mre11, homolog pairing and synapsis, meiotic sister chromatid cohesion, and meiotic progression. We will test the hypothesis that the Rad50 Walker A ATP-binding domain is sufficient for Spo11-mediated DSB initiation. 2. Investigation of the meiotic roles of the Coprinus cinereus adherin protein Rad9. We will examine interactions of conserved and evolutionarily divergent regions of the Rad9 protein in mitotic and meiotic ceils. We will test the hypothesis that at least one, non-cohesion function of Rad9 concerns the ability of the Mre11/Rad50 complex to localize on meiotic chromosomes. We will investigate what functions of this adherin are obviated by the absence of a sister chromatid, and we will isolate new alleles of rad9. 3. Examination of pre-karyogamy events in C. cinereus. These will include the examination of the timing and duration of pre-meiotic S phase, the loading of the meiosis-specific cohesin Rec8, and the requirement for Msh4 and Msh5 in pre-meiotic DNA replication. We will also test the hypothesis that nuclear fusion brings homologs into coordinate regions of the newly diploid cell. 4. Analysis of candidate genes revealed by microarray studies of gene expression during meiosis and after gamma irradiation. We will develop procedures for gene silencing during meiosis, in order to characterize the meiotic functions of genes co-regulated with mre11, rad50, and rad9, and to exploit genomic sequence information as it becomes available.
