Structure and Function of a Eukaryotic Centromere

描述（由申请人提供）：重复染色体的精确分离确保子细胞得到且只有一个染色体的副本。染色体分离错误导致非整倍体，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是肿瘤细胞的特征。因此，分离错误被认为是肿瘤发生的主要原因（Jallepalli and Lengauer, 2001; Yuen et al., 2005）。对染色体分离机制的研究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需的蛋白质方面取得了巨大进展，但对产生关键力的组分的机制和结构以及着丝粒硬度的分子基础仍然知之甚少。我们提出了一种新模型来解释着丝粒中DNA和黏结蛋白的组织（Bloom et al., 2006）。该模型的关键特征是在有丝分裂过程中，着丝点促进着丝粒周围染色质形成十字形，使着丝粒两侧的DNA通过分子内内聚而结合在一起，而染色体臂则在分子间配对。该模型为该领域的主要矛盾提供了解决方案，并将着丝粒DNA的组织与内聚蛋白的分布协调起来。我们的实验方法结合了模型卷积显微镜，活细胞中的单染色体跟踪和着丝点蛋白的高分辨率共定位。我们选择了出芽酵母酿酒酵母来表征力的产生机制和张力元素，这些张力元素存在于着丝点- mt附件的界面上。每个着丝点只有一个微管（MT）附着，并且能够为单分子研究设计设计染色体，这使得出芽酵母成为一个理想的系统。我们的研究将验证这样一个假设，即在整个系统发育中保守的分离的基本单位是出芽酵母附着位点（着丝点），它通过突触的中心周围染色质连接染色体到微管。

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.