EM Studies of Chromosome Organization Assemblies

所有细胞都必须适当地包装和组织它们的DNA才能生存。虽然某种程度的DNA超结构是由超螺旋和小结构蛋白的结合产生的，但被称为凝集素的多蛋白组装负责在更全球的水平上压缩DNA。凝集素复合体是由染色体结构维持蛋白家族的ATPase亚基和一些辅助因子组成的。SMC组分是灵活的、延伸的螺旋卷曲蛋白，将ATP周转与DNA紧凑结合在一起。辅助亚单位的确切功能及其相对于SMC亚基的组织尚不清楚。已经提出了多种机制来解释凝聚素是如何物理凝聚DNA的，尽管这些模型的某些显著特征 这是相互排斥的，而且还没有得到实验验证。这项建议旨在利用生物化学和电子显微镜的组合来：1)确定凝聚素颗粒的整体结构，2)阐明凝聚素颗粒中辅助亚基的作用，以及3)将这些努力的数据应用于测试凝聚素功能的特定模型。为了比较和对比这些组件在不同的细胞王国中的功能，我们将研究酵母和细菌的两个不同的凝集素系统，它们的亚基组成和生化性质不同。为了使这些研究成为可能，我们已经：1)表达和纯化了所有相关的凝集素组件和亚组件，2)开始定义这些复合体与DNA的相互作用，3)从单粒子图像中获得了凝集素复合体的初步重建。到目前为止获得的数据表明 我们具体目标的可行性。

All cells must appropriately package and organize their DNA to survive. While some degree of DNA superstructure is generated by supercoiling and the binding of small architectural proteins, multiprotein assemblies known as condensins are responsible for compacting DNA on a more global level. Condensin complexes are composed of ATPase subunits of the Structural Maintenance of Chromosomes (SMC) protein family as well as several accessory factors. The SMC components are flexible, extended coiled-coil proteins that couple ATP turnover to DNA compaction. The precise function of the accessory subunits and their organization with respect to the SMC subunits are unknown. Multiple mechanisms have been proposed to explain how condensins physically condense DNA, although certain salient features of these models are mutually exclusive and have not been experimentally validated. This proposal aims to employ a combination of biochemistry and electron microscopy to: 1) determine the global architecture of condensin particles, 2) elucidate the role of the accessory subunits in the condensin particle, and 3) apply data from these efforts toward testing specific models of condensin function. In order to compare and contrast the function of these assemblies across different cellular kingdoms, we will study two distinct condensin systems of different subunit composition and biochemical properties from yeast and bacteria. To enable these studies, we have: 1) expressed and purified all pertinent condensin assemblies and subassemblies, 2) begun to define the interaction of these complexes with DNA, and 3) have obtained initial reconstructions of condensin complexes from single particle images. Data obtained to date indicate the feasibility of our specific aims.