Molecular Architecture of the Regional Kinetochore in Fission Yeast

描述(申请人提供)：动粒是一种特殊的蛋白质复合体，它聚集在染色体的特定位置上，称为着丝粒。它们调节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将染色体分开。动粒功能的异常导致非整倍体，这是一种主要的遗传不稳定形式，与许多疾病，特别是癌症有关。这项应用的目的是描绘动粒的结构，并了解动粒在分裂酵母S.pombe中介导染色体分离的机制。裂解酵母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”的极佳模型。地区性动觉中枢特有的问题可以在分子细节上解决，所学到的经验教训可以应用于人类动觉中枢。相比之下，在发芽酵母中发现的“点着丝点动粒”是简单的类型，并结合到一个微管上。我们以前在确定分裂酵母中着丝粒的生化组成方面已经取得了重大进展。我们已经确定了三个主要的动粒复合体，其中包括30个蛋白质。我们还确定了分裂酵母着丝粒的基础是由一系列定位良好的核小体组成，其中三个核小体含有着丝粒特异的组蛋白H3突变体CENP-A(Cnp1p)，对应于与着丝粒结合的三个微管。此外，我们最近在着丝粒中Cnp1-核小体位置的发现表明，Cnp1-核小体占据了优先但灵活的位置的子集。通过测定代表性成分的拷贝数并将其与发芽酵母中的拷贝数进行比较，我们认为裂殖酵母中的区域着丝粒由三个功能单元组成，每个功能单元在结构上类似于一个点着丝粒。重要的是，分裂酵母着丝点没有足够的DAM1复合体拷贝来组装16-聚环，这一结构对于DAM1‘S在萌芽酵母中作为染色体运动和微管解聚的耦合器起着至关重要的作用。在接下来的资助期，我们将通过在分子水平上测试“重复单位”模型，进一步研究动粒在分裂酵母中的分子组装。该模型假定裂殖酵母中的区域着丝粒由多个单元组成，每个单元能够与一个MT结合。我们将确定一个单位的染色质基础是什么，以及如何安排多个单位形成一个完整的动粒(目标1)。我们将描述动粒单元的结构特征和动粒组件之间的全面功能联系(目标2)。我们还将研究分裂酵母Dam1的功能和机制，它也参与耦合染色体运动和微管解聚，但在小于16-聚环的多聚体组装中发挥作用(Aim3)。公共卫生相关性：该项目研究有丝分裂机制的关键组成部分着丝粒是如何组装的，并在调节染色体分离中发挥作用。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量的异常--称为非整倍体。非整倍体与许多人类疾病有关，尤其与肿瘤的发生密切相关。该项目将在单细胞生物体--分裂酵母S.pombe中进行，这是研究人类中发现的区域动点类型的一个极好的模型。

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.