Biochemical Analysis of Sister Chromatid Cohesion

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DESCRIPTION (APPLICANT'S ABSTRACT): Sister chromatid cohesion is an essential cellular process that ensures the faithful segregation of genetic information during mitosis and meiosis. The long-term goal of this project is to understand the biochemical basis of this process by using cell-free extracts derived from Xenopus laevis (African toad) eggs and mammalian tissue culture cells. An emphasis will be made on the structural and functional characterization of cohesin, a highly conserved protein complex that plays a key role in establishing and maintaining sister chromatid cohesion. The cohesin complex is composed of two SMC (structural maintenance of chromosomes) subunits and at least two non-S MC subunits, and is predicted to act as part of the molecular "glue" that holds two sister chromatids together. In this proposal, (1) Biochemical and structural approaches will be combined to understand the molecular mechanisms of action of the cohesin complex. (2) Cell cycle-dependent interactions between cohesin and chromatin will be reconstituted in vitro and the molecular basis of loading and unloading processes will be determined. (3) Vertebrate homologs of BimD and Scc2, two proteins implicated genetically in sister chromatid cohesion, will be identified and characterized in the cell-free extracts and in tissue culture cells. (4) A novel in vitro assay will be developed to determine how sister chromatid cohesion is functionally coupled to DNA replication. The information obtained from this work will ultimately contribute to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocations, are tightly associated with tumor development and birth defects.