Analysis of Meiotic Chromosome Synapsis in Yeast

描述(申请人提供)：减数分裂是一种进化上保守的细胞过程，通过这种过程，生殖细胞的染色体数量减少了一半，使得每个配子只接收每个染色体对中的一个成员。染色体的正确分离依赖于一系列紧密协调的事件，涉及到染色体的突触和重组。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(AEs)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。这项建议的重点是通过分析萌芽酵母中三个关键的减数分裂特定的AE组分，HOP1，Red1和MEK1，来了解AE功能如何促进同源染色体分离。 MEK1是一种丝氨酸/苏氨酸蛋白激酶。通过扩大MEK1的ATP结合口袋，我们创造了MEK1的模拟敏感(AS)等位基因。MEK1-AS的激酶活性可以被小分子特异性地抑制；此外，使用放大的ATP衍生物进行的激酶分析允许检测MEK1-AS的特定底物。利用MEK1-AS等位基因，我们已经证明了Mek1激酶活性是形成双链断裂(DSB)以及姐妹染色单体交换障碍所必需的，但不是修复所必需的。与文献相反，MEK1不能磷酸化Red 1；相反，Hop 1 Red 1复合体是MEK1激酶活性所必需的。这些观察结果导致了一个新的HOP1/Red1/MEK1功能在减数分裂中的作用机制的模型，我们建议在这项研究中进行测试。此外，我们计划利用MEK1-AS等位基因在遗传和生化筛查中的独特性质来鉴定MEK1的直接底物。 HOP 1是一种磷酸化蛋白，其修饰依赖于CDC7蛋白的活性。CDC7是一种进化上保守的激酶，对营养细胞中的DNA复制是必不可少的。然而，在减数分裂中，CDC7突变体在前减数分裂S之后、重组和染色体联会之前停止。利用CDC7的AS等位基因，我们计划测试CDC7减数分裂停滞是由于未能直接磷酸化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.