Biochemical Analysis of Sister Chromatid Cohesion

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DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the molecular mechanisms of chromosome cohesion and segregation. Central to this process are members of an evolutionary conserved family of chromosomal ATPase, known as the structural maintenance of chromosomes (SMC) family. SMC proteins form dimers, which adopt a unique two-armed structure with an ATP-binding "head" domain at the distal end of each arm. In eukaryotes, an SMC protein complex known as cohesin plays a key role in sister chromatid cohesion in mitosis and meiosis. In this proposal, multi-disciplinary approaches will be taken to understand how SMC proteins work at a mechanistic level in vitro and how their functions are regulated in vivo. First, the bacterial SMC protein complex from Bacillus subtilis will be used as a model system for understanding the basic: action of SMC proteins. An innovative set of functional assays, combined with extensive site-directed mutagenesis, will be employed to address how the mechanical cycle of SMC might be coupled to its catalytic cycle. These biochemical studies will be complemented by other approaches including single-DNA-molecule nanomanipulation and protein crystallization. Second, sub- and holo-complexes of cohesin will be reconstituted from its recombinant subunits, and their activities will be characterized in both purified systems and Xenopus egg cell-free extracts. Mutant forms of the complexes will be constructed to determine how the mechanochemical cycle of cohesin participates in sister chromatid cohesion and its release. Finally, regulation of cohesin's loading and stabilization will be studied in Xenopus egg extract:; and mammalian tissue culture cells. A major emphasis will be made on the role of 3 different classes of regulators (Scc2, Pds5 and Sgo) in these processes. The impacts of Scc2 and Pds5 on the ATPase cycle of cohesin will also be determined. The information obtained from this work will ultimately lead to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocation, are tightly associated with tumor development and birth defects.