EM Studies of Chromosome Organization Assemblies

University Of Calif-Lawrenc Berkeley Lab, Berkeley CA

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Abstract

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.