A Molecular View of Chromosome Recombination & Segregation in Eukaryotic Meiosis

描述(由申请人提供)： 减数分裂是一种特殊的细胞分裂程序，它在有性繁殖的有机体中产生配子。减数分裂的第一阶段，称为减数分裂I，独特地涉及同源染色体的联合、程序性重组和最终的分离。虽然从遗传学和细胞学的角度很好地理解了这一过程，但我们对减数分裂特有的细胞机制如何能够在3D空间组织和操纵减数分裂染色体以调节其适当分离的理解仍然是一个谜。这一领域的研究意义重大，因为减数分裂I期染色体分离的错误导致了绝大多数非整倍体，即后代中额外或缺失的染色体，这种情况发生在超过一半的人类卵母细胞和5%-10%的临床公认的妊娠中。因此，非整倍体是流产和智力低下的主要遗传原因(例如，由21号染色体三体引起的唐氏综合症)。减数分裂I中染色体分离错误的潜在原因尚不清楚，进一步确定这些原因将需要详细了解减数分裂特有的染色体分离机制的分子机制。在这里，我们建议研究三组减数分裂染色体相关蛋白，它们对减数分裂I中染色体组织和物理操作的不同方面至关重要。我们的方法结合了纯化蛋白质和复合体的体外重组、这些复合体的三维结构分析和有针对性的遗传分析，以测试旨在破坏这些蛋白质结构和相互作用特定方面的突变体。我们将首先研究酿酒酵母单核蛋白复合体，它在减数分裂I中结合染色体的动点，并将其附着在纺锤体微管上，以使同源染色体能够正确定位和分离。我们将确定单链蛋白复合体的结构，并在基因分析中使用工程蛋白结构来测试它是否直接与姐妹动素中心相互作用。 它们附着在单个微管上。接下来，我们将研究保守的染色体相关蛋白Hop1，这是每条染色体围绕其组织的蛋白质“轴”的一个组成部分。我们将研究Hop1的S保守的Horma结构域，该结构域与纺锤体检查点蛋白MAD2共享的一个信号域，在调节同源减数分裂间的减数分裂重组和减数分裂特异性检查点监控重组中的作用。最后，我们将研究联会复合体，这是一种在减数分裂重组过程中将同系物连接在一起的重要聚合组装。由于对该复合体的结构或功能知之甚少，我们将研究关键的酿酒酵母联会复合体蛋白ZIP1的结构域结构、蛋白质-蛋白质相互作用和自组装决定因素。结合起来，这项工作将开始为减数分裂I中同源染色体重组和分离背后的大分子结构和相互作用提供更准确的图景。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Meiosis is a specialized cell division program that gives rise to gametes in sexually reproducing organisms. The first stage of meiosis, called meiosis I, uniquely involves the association, programmed recombination, and eventual segregation of homologous chromosomes. While this process is well understood from a genetic and cytological standpoint, our understanding of how the meiosis-specific cellular machinery is able to organize and manipulate meiotic chromosomes in 3D space to mediate their proper segregation remains a mystery. This area of study is significant, as errors in meiosis I chromosome segregation account for the vast majority of aneuploidies, extra or missing chromosomes in offspring, that occur in over half of human oocytes and 5-10% of clinically recognized pregnancies. As such, aneuploidy is the leading genetic cause of miscarriage and of mental retardation (e.g. Down syndrome, caused by trisomy of chromosome 21). The underlying causes of chromosome segregation errors in meiosis I are not well understood, and further progress toward identifying these causes will require a detailed understanding of the molecular mechanisms of meiosis-specific chromosome segregation machinery. Here, we propose to study three sets of meiotic chromosome-associated proteins that are critical for different aspects of chromosomes' organization and physical manipulation in meiosis I. Our approach combines in vitro reconstitution of purified proteins and complexes, 3D structural analysis of these complexes, and targeted genetic assays to test mutants designed to disrupt specific aspects of these proteins' structures and interactions. We will first study the S. cerevisiae monopolin complex, which binds chromosomes' kinetochores in meiosis I and modifies their attachments to spindle microtubules, to enable the proper orientation and segregation of homologous chromosomes. We will determine the architecture of the monopolin complex, and use engineered protein constructs in genetic assays to test whether it directly cross-links sister kinetochores to mediate their attachment to a single microtubule. Next, we will study the conserved chromosome-associated protein Hop1, a component of the proteinaceous "axis" about which each chromosome is organized. We will examine the roles of Hop1's conserved HORMA domain, a signaling domain shared with the spindle checkpoint protein Mad2, in regulating inter-homolog meiotic recombination, and in a meiosis-specific checkpoint monitoring recombination. Finally, we will study the synaptonemal complex, an essential polymeric assembly that links homologs together during meiotic recombination. As very little is known about the architecture of this complex or its functions, we will study the domain structure, protein-protein interactions, and self-assembly determinants of the key S. cerevisiae synaptonemal complex protein Zip1. Combined, this work will begin to provide a more accurate picture of the macromolecular structures and interactions underlying homologous chromosome recombination and segregation in meiosis I.