Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis.

Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the "ZMM" genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis.

Cdc7-Dbf4 is a gene-specific regulator of meiotic transcription in yeast.

Meiosis divides the chromosome number of the cell in half by having two rounds of chromosome segregation follow a single round of chromosome duplication. The first meiotic division is unique in that homologous pairs of sister chromatids segregate to opposite poles. Recent work in budding and fission yeast has shown that the cell cycle kinase, Cdc7-Dbf4, is required for many meiosis-specific chromosomal functions necessary for proper disjunction at meiosis I. This work reveals another role for Cdc7 in meiosis as a gene-specific regulator of the global transcription factor, Ndt80, which is required for exit from pachytene and entry into the meiotic divisions in budding yeast. Cdc7-Dbf4 promotes NDT80 transcription by relieving repression mediated by a complex of Sum1, Rfm1, and a histone deacetylase, Hst1. Sum1 exhibits meiosis-specific Cdc7-dependent phosphorylation, and mass spectrometry analysis reveals a dynamic and complex pattern of phosphorylation events, including four constitutive cyclin-dependent kinase (Cdk1) sites and 11 meiosis-specific Cdc7-Dbf4-dependent sites. Analysis of various phosphorylation site mutants suggests that Cdc7 functions with both Cdk1 and the meiosis-specific kinase Ime2 to control this critical transition point during meiosis.

Using the semi-synthetic epitope system to identify direct substrates of the meiosis-specific budding yeast kinase, Mek1.

Recent studies have shown that the meiosis-specific kinase, Mek1, plays a key role in promoting recombination between homologous chromosomes during meiosis in budding yeast by suppressing recombination between sister chromatids, as well as playing a role in the meiotic recombination checkpoint. Understanding how Mek1 regulates recombination requires the identification of direct substrates of the kinase. We have applied the semi-synthetic epitope method developed by Shokat and colleagues to Mek1. This method uses an analog-sensitive version of Mek1, GST-Mek1-as, in conjunction with an ATPγS analog, for kinase assays that detect only those proteins that are directly phosphorylated by Mek1. This method may be applicable to any kinase for which an analog-sensitive version is available. In addition, it provides a non-radioactive alternative for kinase assays with wild-type kinases.

Cdc7-Dbf4 regulates NDT80 transcription as well as reductional segregation during budding yeast meiosis.

In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induction of genes involved in exit from pachytene, meiotic progression, and spore formation. Second, Cdc7 is necessary for recruitment of monopolin to sister kinetochores, and it is necessary for the reductional segregation occurring at meiosis I. The use of the same kinase to regulate several distinct meiosis-specific processes may be important for the coordination of these processes during meiosis.