Molecular Architecture of the Regional Kinetochore in Fission Yeast

描述（由申请人提供）：着丝点是一种特殊的蛋白质复合物，组装在称为着丝粒的染色体的特定位点上。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过程中分离染色体。着丝点的异常功能导致非整倍体，这是一种与许多疾病，特别是癌症有关的主要遗传不稳定性形式。本应用程序的目的是描述着丝点的结构，并了解着丝点介导分裂酵母S.pombe染色体分离的机制。分裂酵母的着丝点是研究大多数真核生物中发现的与多个微管结合的“区域着丝点”的一个很好的模型。区域着丝点特有的问题可以在分子细节上解决，所吸取的经验教训可以应用于人类着丝点。相比之下，在出芽酵母酿酒酵母中发现的“点着丝点”是简单的类型，与一个微管结合。我们以前在确定裂变酵母中着丝点的生化组成方面取得了重大进展。我们已经确定了三种主要的着丝点复合物，包括30种蛋白质。我们还确定了分裂酵母着丝点的着丝基础是由一系列位置良好的核小体组成的，其中三个包含着丝点特异性组蛋白H3变体Cenp-A (Cnp1p)，对应于与着丝点结合的三个微管。此外，我们最近在着丝粒中cnp1核小体位置的发现表明，cnp1核小体占据了一个优先但灵活的位置子集。通过确定代表性成分的拷贝数，并将其与出芽酵母中的拷贝数进行比较，我们提出裂变酵母中的区域着丝点由三个功能单元组成，每个功能单元在结构上与点着丝点相似。重要的是，裂变酵母的着丝点没有足够的Dam1复合体拷贝来组装一个16-mer环，这个结构对于Dam1在出芽酵母中作为染色体运动耦合器和微管解聚的功能至关重要。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，进一步研究裂变酵母中着丝点的分子组装。该模型假设分裂酵母中的区域着丝点由多个单元组成，每个单元都能够与一个MT结合。我们将确定一个单元的染色质基础由什么组成，以及多个单元如何排列以形成一个完整的着丝点（目标1）。我们将描述着丝点单元的结构特征和着丝点组成部分之间的综合功能联系（目标2）。我们还将研究裂变酵母Dam1的功能和机制，它也参与偶联染色体运动和微管解聚，但在小于16聚环的多聚体组装中起作用（Aim3）。公共卫生相关性：本项目研究着丝点是有丝分裂机制的关键组成部分，如何组装并在介导染色体分离中起作用。这一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-称为非整倍体。非整倍体与许多人类疾病有关，特别是与肿瘤发生密切相关。该项目将在一种单细胞生物中进行，即裂变酵母S.pombe，它是研究人类中发现的区域着丝点的绝佳模型。

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.