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，但被称为凝聚素的多蛋白组装体负责在更全局的水平上压缩DNA。凝聚素复合物是由染色体结构维持蛋白家族的ATP酶亚基以及一些辅助因子组成的。SMC组分是柔性的、延伸的卷曲螺旋蛋白，其将ATP周转与DNA压缩偶联。附属亚单位的确切功能及其相对于SMC亚单位的组织尚不清楚。已经提出了多种机制来解释凝聚素如何物理地凝聚DNA，尽管这些模型的某些显著特征是不确定的。 相互排斥，并且没有经过实验验证。该提案旨在采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的功能，我们将研究两个不同的凝聚素系统的不同亚基组成和生化特性的酵母和细菌。为了使这些研究成为可能，我们已经：1）表达并纯化了所有相关的凝聚素组装体和DNA，2）开始定义这些复合物与DNA的相互作用，3）已经从单粒子图像获得了凝聚素复合物的初步重建。迄今为止获得的数据表明， 我们的具体目标的可行性。

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.