| The pachytene checkpoint monitors the status of meiotic recombination, a process that is essential for correct homolog segregation at meiosis I. This checkpoint operates in the zip1 mutant, which arrests at the pachytene stage of meiosis with defects in recombination and synaptonemal complex (SC) formation. To understand the molecular details of this checkpoint, a genetic screen was carried out to search for mutations that allow zip1 to sporulate. The screen identified transposon insertions in RAD24, RED1, SWE1, MSC3, ELF1, MEK1, HSP104, PCH2, DDC1 and SIR4.; Ddc1 and its interacting partners (Rad17 and Mec3) are presumed to form a donut-shaped structure that clamps around DNA in a manner analogous to proliferating-cell nuclear antigen (PCNA). This structural similarity to PCNA makes the prediction that Ddc1 associates with damaged DNA. Indeed, Ddc1 localizes to sites of double-strand breaks (DSBs) on meiotic chromosomes. Ddc1 interacts with Rad24, which in turn associates with recombination proteins, suggesting formation of a multi-protein complex at the sites of DSBs. Ddc1's association with DSBs is promoted by the presence of single-stranded (ss) DNA, implying that DSBs with ss DNA are the substrate sensed by the checkpoint. Ddc1 is a phosphoprotein. Ddc1 phosphorylation depends on the meiosis-specific Mek1 kinase; in turn, Ddc1 is important for anchoring Mek1 to the sites of DNA damage and facilitating Mek1-dependent phosphorylation of Red1. This places Ddc1 in a positive feedback loop with respect to Mek1. This feedback loop between Ddc1 and Mek1 might be important for amplification and maintenance of the checkpoint signal.; In an attempt to position Pch2 with respect to Ddc1 in the checkpoint pathway, genetic epistasis tests were carried out. The results indicate that Ddc1 and Pch2 function in parallel pathways. Genetic evidence suggests that Ddc1 is specific to a checkpoint pathway monitoring DSBs bearing ss DNA, while Pch2 is specific to a checkpoint pathway monitoring blunt DSBs. Work is underway to test whether Pch2 acts in the same pathway as Tel1 and, if so, to position Pch2 with respect to Tel1.; Sir4 is a transcriptional silencer that operates at the silent cassettes of mating type information and at telomeres. A non-null allele that creates a C-terminal truncation of the Sir4 protein, designated Sir4-t, disrupts the checkpoint. Sir4 localizes to telomeres and to the nucleolus during meiosis, while the truncated form of Sir4 fails to localize to the nucleolus. (Abstract shortened by UMI.)... |
