Molecular Architecture of the Regional Kinetochore in Fission Yeast

描述（由申请人提供）：动粒是在染色体的特定位点（称为着丝粒）上组装的特化蛋白质复合物。它们介导染色体和纺锤体微管之间的相互作用，纺锤体微管在有丝分裂期间分离染色体。动粒的异常功能导致非整倍性，这是与许多疾病，特别是癌症有关的遗传不稳定性的主要形式。本申请的目标是描绘的动粒的架构，并了解的机制，其中动粒介导的染色体分离的裂殖酵母粟酒裂殖酵母。裂殖酵母动粒是研究大多数真核生物中发现的“区域动粒”的极好模型，其结合到多个微管。区域动粒所特有的问题可以在分子细节上得到解决，所学到的教训可以应用于人类动粒。相比之下，在芽殖酵母酿酒酵母中发现的“点动粒”是简单类型的，并且结合到一个微管。我们以前已经取得了重大进展，在确定分裂酵母中动粒的生化组成。我们已经确定了三个主要的动粒复合物，包括三十种蛋白质。我们还确定了一个分裂酵母动粒的着丝粒基础是由一系列定位良好的核小体组成的，其中三个含有着丝粒特异性组蛋白H3变体Cenp-A（Cnp 1 p），对应于三个微管结合到动粒。此外，我们最近在Cnp 1-核小体的位置在着丝粒的发现表明，Cnp 1-核小体占据了一个子集的首选，但灵活的位置。通过确定代表性组分的拷贝数，并将它们与芽殖酵母中的那些进行比较，我们提出，裂变酵母中的区域动粒由三个功能单元组成，每个功能单元在结构上与点动粒相似。重要的是，裂殖酵母动粒不具有足够的Dam 1复合物的拷贝来组装16-mer环，这是一种对于Dam 1作为芽殖酵母中染色体运动和微管解聚的耦合剂的功能至关重要的结构。在下一个资助期内，我们将通过在分子水平上测试“重复单元”模型，进一步研究分裂酵母中动粒的分子组装。该模型假设，在裂变酵母的区域动粒是由多个单位，每个单位能够结合到一个MT。我们将确定一个单位的染色质基础是由什么组成的，以及多个单位是如何排列形成一个完整的动粒的（目标1）。我们将描述动粒单位的结构特征和动粒组成部分之间的全面功能联系（目标2）。我们还将研究裂殖酵母Dam 1的功能和机制，Dam 1也参与耦合染色体运动和微管解聚，但在小于16- mer环的多聚体组装体（Aim 3）中起作用。公共卫生相关性：本计画主要研究有丝分裂机制中的重要组成部分动粒如何组装，并在介导染色体分离中发挥功能。这些知识将具有重要的治疗意义，因为染色体分离的缺陷直接导致染色体数目的异常-称为非整倍性。非整倍性与许多人类疾病有关，特别是与肿瘤发生密切相关。该项目将在单细胞生物体中进行，裂变酵母S.pombe，这是研究人类中发现的区域动粒类型的极好模型。

DESCRIPTION (provided by applicant): Kinetochores are specialized protein complexes that are assembled on specific loci of chromosomes named centromeres. They mediate the interaction between chromosomes and the spindle microtubules, which separate chromosomes during mitosis. Aberrant function of kinetochore leads to aneuploidy, a major form of genetic instability implicated with a number of diseases, in particular, cancers. The goal of this application is to delineate the architecture of the kinetochore and to understand the mechanisms by which kinetochores mediate chromosome segregation in the fission yeast S.pombe. The fission yeast kinetochore is an excellent model for the study of "regional kinetochores" found in most eukaryotes, which bind to multiple microtubules. Questions unique to regional kinetochores can be addressed in molecular detail and the lessons learnt can be applied to human kinetochores. In contrast, the "point kinetochore" found in the budding yeast S.cerevisiae is the simple type and binds to one microtubule. We previously have made major progress in determining the biochemical composition of the kinetochore in the fission yeast. We have identified three major kinetochore complexes including thirty proteins. We have also determined that the centromeric foundation of a fission yeast kinetochore is composed of an array of well-positioned nucleosomes, among which three contain centromere-specific histone H3 variant Cenp-A (Cnp1p), corresponding to three microtubules bound to the kinetochore. Furthermore, our recent finding in Cnp1-nucleosome positions in the centromere indicates that Cnp1-nucleosomes occupy a subset of preferred but flexible positions. By determining the copy numbers of the representative components and comparing them to those in the budding yeast, we propose that a regional kinetochore in fission yeast is comprised of three functional units, each is architecturally similar to a point kinetochore. Importantly, fission yeast kinetochores do not possess sufficient copies of the Dam1 complex to assemble a 16-mer ring, a structure crucial for the Dam1's function as a coupler of chromosome movement and microtubule depolymerization in the budding yeast. In the next funding period, we will further investigate the molecular assembly of kinetochores in the fission yeast by testing a "repeat unit" model at the molecular level. The model postulates that the regional kinetochore in the fission yeast is comprised of multiple units, each unit is capable of binding to one MT. We will determine what comprises of the chromatin foundation of one unit and how multiple units are arranged to form a whole kinetochore (Aim 1). We will delineate the architectural features of the kinetochore unit and the comprehensive functional connections among the kinetochore components (Aim 2). We will also investigate the functions and the mechanism of fission yeast Dam1, which is also involved in coupling chromosome movement and microtubule depolymierization, but functions in the multimeric assemblies smaller than a 16- mer ring (Aim3). PUBLIC HEALTH RELEVANCE: This project studies how a kinetochore, a crucial component of the mitosis machinery, is assembled and functions in mediating chromosome segregation. This knowledge will have important therapeutic implications, since defects in chromosome segregation directly lead to abnormality in chromosome numbers - called aneuploidy. Aneuploidy is implicated in many human diseases, and in particular, is closely involved in tumorigenesis. This project will be conducted in a single cell organism, the fission yeast S.pombe, which is an excellent model for the study of the regional kinetochores, the type found in humans.