Meiosis is a highly conserved specialized cell division that occurs in many organisms, including budding yeast and mammals. Meiosis divides the chromosome number of the cell in half to create gametes for sexual reproduction. A single round of chromosome duplication is followed by two rounds of chromosome segregation, Meiosis I (homologs segregate) and Meiosis II (sister chromatids segregate). Proper segregation at Meiosis I requires that homologs are connected by both crossovers and sister chromatid cohesion. Crossovers are formed by the repair of double strand breaks (DSBs) preferentially by the homolog. The choice of repair template is determined at the time of strand invasion, which is mediated by two recombinases, Rad51 and the meiosis-specific Dmc1. Rad51 is necessary for Dmc1 to function properly but its strand exchange activity is inhibited both by Dmc1 and Mek1, a meiosis-specific kinase, which is activated by DSBs. Mek1 suppresses interaction between Rad51 and its accessory factor Rad54 in two ways. First, phosphorylation of Rad54 lowers its affinity for Rad51. Second, phosphorylation stabilizes Hed1, a meiosis-specific protein that binds to Rad51 and excludes Rad54. Although RAD54 is not required for wild-type levels of interhomolog recombination, rad54Δ diploids exhibit decreased sporulation and spore viability, indicating the presence of unrepaired DSBs. My thesis tested the idea that Mek1 kinase activity is down-regulated after interhomolog recombination to allow Rad51-mediated repair of any remaining DSBs.
Meiotic recombination occurs in the context of a proteinaecous structure called the synaptonemal complex (SC). The SC is formed when sister chromatids condense along protein cores called axial elements (AEs) comprised of the meiosis-specific proteins, Hop1, Red1 and Rec8. AEs are brought together by interhomolog recombination, which creates stable connections and the gluing together of the AEs by the insertion of the transverse filament protein, Zip1, in a process called synapsis. Pachynema is the stage of meiotic prophase in which chromosomes are fully synapsed and where interhomolog recombination has proceeded to the double Holliday junction (dHJ) stage.
Meiotic progression requires transcription factor NDT80, a middle meiosis transcription factor required to express >200 genes, including the polo-like kinase, CDC5 (required for Holliday junction resolution and SC disassembly) and CLB1 (required for meiotic progression). Diploids deleted for NDT80 arrest in pachynema with unresolved dHJs. I used an inducible version of NDT80 (NDT80-IN ) to separate prophase into two phases: pre-NDT80, when interhomolog recombination occurs and post-NDT80, when it is proposed that inactivation of Mek1 allows intersister recombination to repair residual DSBs. RAD54 is sufficient to function after interhomolog recombination, as inducing both RAD54 and NDT80 simultaneously rescues the spore inviability defects observed in NDT80-IN rad54Δ diploids. Using an antibody specific for phosphorylated Hed1 as an indicator of Mek1 kinase activity, I showed that Mek1 is constitutively active in ndt80-arrested cells and that induction of NDT80 is sufficient to abolish Mek1 activity. Furthermore, inactivation of Mek1 by Ndt80 can occur in the absence of interhomolog strand invasion and synapsis. Mek1 inactivation correlates with the appearance of CDC5 and the degradation of Red1. My work demonstrates that the sole target of NDT80 responsible for inactivating Mek1 is CDC5.
Unrepaired DSBs trigger the meiotic recombination checkpoint resulting in prophase arrest, which requires Mek1 and works by sequestering Ndt80 in the cytoplasm. Mek1 also delays meiotic progression in wild-type cells, likely through inactivation of Ndt80. My work shows that Ndt80 in turn negatively regulates Mek1. Based on my observations, as well as published work showing that synapsis results in the removal of Mek1 from chromosomes, I propose that recombination and meiotic progression are coordinated by regulation of Mek1.
|Advisor:||Hollingsworth, Nancy M.|
|Commitee:||Futcher, Bruce, Hochwagen, Andreas, Luk, Ed, Neiman, Aaron|
|School:||State University of New York at Stony Brook|
|Department:||Molecular and Cellular Biology|
|School Location:||United States -- New York|
|Source:||DAI-B 78/03(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Genetics, Cellular biology|
|Keywords:||Meiosis, Mek1, Rad51, Yeast|
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