Analysis of Meiotic Chromosome Synapsis in Yeast

描述（由申请人提供）：减数分裂是一种进化上保守的细胞过程，生殖细胞的染色体数目减少一半，使得每个配子仅接受每个染色体对的一个成员。染色体的正确分离依赖于一系列紧密协调的事件，包括染色体联会和重组。遗传学和细胞学研究表明，姐妹染色单体沿着蛋白质核心凝聚形成轴向元件（AE）是促进同源染色体在第一次减数分裂时正确分离的重要特征。该提案的重点是通过分析芽殖酵母中三个关键的减数分裂特异性AE组分HOP 1、RED 1和MEK 1来了解AE功能如何促进同源染色体分离。 Mek 1是一种丝氨酸/苏氨酸蛋白激酶。通过扩大ATP结合口袋的Mec 1，我们已经创建了一个类似物敏感（作为）等位基因的Mek 1。Mek 1-as的激酶活性可以被小分子特异性抑制;此外，使用放大的ATP衍生物的激酶测定允许检测Mek 1-as的特异性底物。使用的mek 1-作为等位基因，我们已经表明，Mekl激酶活性的形成，但不修复，双链断裂（DSB），以及姐妹染色单体交换的障碍。与文献相反，Mek 1不磷酸化Red 1;相反，Hop 1 Red 1复合物是Mek 1激酶活性所必需的。这些观察结果为HOP 1/RED 1/MEK 1在减数分裂中的功能机制提供了一个新的模型，我们建议在本研究中进行测试。此外，我们计划利用独特的属性的mek 1-作为等位基因的遗传和生化筛选，以确定直接底物的Mek 1。 Hop 1是一种磷蛋白，其修饰依赖于Cdc 7激酶活性。Cdc 7是一种进化上保守的激酶，对营养细胞中的DNA复制至关重要。然而，在减数分裂中，cdc 7突变体在减数分裂前S后，重组和染色体联会之前停止。使用CDC 7的作为等位基因，我们计划测试cdc 7减数分裂停滞是由于不能直接磷酸化Hop 1的假设。

DESCRIPTION (provided by applicant): Meiosis is an evolutionarily conserved cellular process by which the chromosome number of germ cells is reduced in half such that each gamete receives only one member of each chromosome pair. The correct segregation of chromosomes is dependent upon a tightly coordinated series of events involving chromosome synapsis and recombination. Genetic and cytological studies have shown that condensation of sister chromatids along protein cores to form axial elements (AEs) is an important feature in promoting proper segregation of homologous chromosomes at the first meiotic division. The focus of this proposal is to understand how AE function promotes homologous chromosome segregation by analyzing three key meiosis specific AE components in budding yeast, HOP1, RED1, and MEK1. Mek1 is a serine/threonine protein kinase. By enlarging the ATP binding pocket of Mec1, we have created an analog-sensitive (as) allele of Mek1. The kinase activity of Mek1-as can be specifically inhibited by small molecules; in addition, kinase assays using enlarged derivatives of ATP allow the detection of specific substrates of Mek1-as. Using the mek1-as allele, we have shown that Mekl kinase activity is required for the formation, but not repair, of double strand breaks (DSBs) as well as for the barrier to sister chromatid exchange. Contrary to the literature, Mek1 does not phosphorylate Red 1; instead Hop 1Red 1 complexes are necessary for Mek1 kinase activity. These observations have led to a new model for the mechanism of HOP1/RED1/MEK1 function in meiosis that we propose to test in this grant. Furthermore, we plan to exploit the unique properties of the mek1-as allele in genetic and biochemical screens to identify direct substrates of Mek1. Hop 1 is a phosphoprotein whose modification is dependent upon Cdc7 kinase activity. Cdc7 is an evolutionarily conserved kinase that is essential for DNA replication in vegetative cells. In meiosis, however, cdc7 mutants arrest after premeiotic S, before recombination and chromosome synapsis. Using an as allele of CDC7, we plan to test the hypothesis that the cdc7 meiotic arrest results from a failure to directly phosphorylate Hop 1.