Structure and Function of a Eukaryotic Centromere

描述（由申请人提供）：重复染色体的准确分离确保子细胞获得每条染色体的一个且仅一个拷贝。染色体分离的错误导致非整倍体，并对人类健康造成严重后果。不正确的染色体数目和染色体不稳定性是肿瘤细胞的标志。因此，分离错误被认为是肿瘤发生的主要原因（Jallepalli和Lengauer，2001; Yuen等人，2005年）的报告。研究染色体分离的机制对于理解可能导致错误的过程至关重要。近年来，在鉴定染色体运动和分离所必需的蛋白质方面取得了巨大进展，但对关键力产生组分的机制和结构以及着丝粒刚度的分子基础仍然知之甚少。我们已经提出了一个新的模型，用于着丝粒中着丝粒周围DNA和粘着蛋白的组织（Bloom et al.，2006年）。该模型的关键特征是着丝粒促进有丝分裂中臂间染色质组织成十字形，使得着丝粒侧翼DNA通过分子内凝聚力保持在一起，而染色体臂在分子间配对。该模型提供了一个解决方案，在该领域的主要矛盾，并调和着丝粒DNA的组织与粘着蛋白的分布。我们的实验方法结合了联合收割机模型卷积显微镜，活细胞中的单染色体跟踪和动粒蛋白的高分辨率共定位。我们选择了芽殖酵母S。cerevisiae来表征位于kinetochore-MT附件界面处的力产生机制和张力元件。每个着丝粒只有一个微管（MT）附着，并且能够设计用于单分子研究的设计染色体，这使得芽殖酵母成为理想的系统。我们的研究将检验这样一个假设，即在整个染色体发生过程中保守的分离的基本单位是芽殖酵母附着位点（动粒），它通过突触的臂间染色质将染色体连接到微管。

DESCRIPTION (provided by applicant): Accurate segregation of duplicated chromosomes ensures that daughter cells get one and only one copy of each chromosome. Errors in chromosome segregation result in aneuploidy and have severe consequences on human health. Incorrect chromosome number and chromosomal instability are hallmarks of tumor cells. Hence, segregation errors are thought to be a major cause of tumorigenesis (Jallepalli and Lengauer, 2001; Yuen et al., 2005). A study of the mechanism of chromosome segregation is essential to understand the processes that can lead to errors. Tremendous progress has been made in recent years in identifying the proteins necessary for chromosome movement and segregation, but the mechanism and structure of critical force generating components and the molecular basis of centromere stiffness remain poorly understood. We have proposed a new model for the organization of pericentric DNA and cohesin in the centromere (Bloom et al., 2006). The key feature of the model is that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA is held together via intramolecular cohesion, while chromosome arms are paired inter-molecularly. The model provides a solution to the major paradoxes in the field and reconciles the organization of centromere DNA with the distribution of cohesin. Our experimental approaches combine model convolution microscopy, single chromosome tracking in living cells, and high resolution colocalization of kinetochore proteins. We have chosen budding yeast S. cerevisiae to characterize the force producing mechanisms and tension elements that reside at the interface of kinetochore-MT attachments. Only one microtubule (MT) attachment per kinetochore and the ability to engineer designer chromosomes for single-molecule studies makes budding yeast an ideal system. Our studies will test the hypothesis that the fundamental unit of segregation conserved throughout phylogeny is the budding yeast attachment site (kinetochore) that links the chromosome via synapsed pericentric chromatin to the microtubule.