BIOCHEMICAL ANALYSIS OF MITOTIC CHROMOSOME CONDENSATION

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DESCRIPTION (provided by applicant): Chromosome condensation 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 how the higher order assembly of mitotic chromosomes is achieved at a molecular level and how the process is precisely regulated during the eukaryotic cell cycle. A major emphasis will be made on the structural and functional characterization of condensins, a class of multi-protein complexes that play fundamental roles in chromosome condensation from yeast to humans. The canonical condensin complex (hereafter referred to as condensin I) was originally discovered by the applicant's laboratory from Xenopus laevis (African toad) egg extracts. Most recently, the same laboratory has identified a second type of condensin (condensin II) from animal cells, making it possible to probe the molecular architecture of mitotic chromosomes in greater details. In this proposal, multi-disciplinary approaches will be taken to understand how the two condensing complexes collaborate to build up metaphase chromosomes and to support their faithful segregation in anaphase. First, condensin II will be purified from human cell nuclear extracts and Xenopus egg extracts, and its subunit composition will be fully described. The biochemical activities of this new complex will be assayed in vitro and its role in chromosome assembly will be tested in Xenopus egg extracts. Second, the localization of condensin I and condensin II on mitotic chromosomes will be determined at high resolution by both light and electron microscopy. A pseudo-genetic approach involving small interference RNAs (siRNAs) will be combined to understand the differential contributions of the two condensin complexes to chromosome architecture and segregation in vivo. Third, the sub- and holo-complexes of condensins will be reconstituted from recombinant subunits, and their mutant forms will be constructed. The recombinant complexes will then be used to understand how the mechanical cycle of condensins is coupled with their catalytic cycle. Moreover, the biochemical assays will be complemented by biophysical approaches including a DNA nanomanipulation technique. The information obtained from this work will ultimately lead to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocations, are tightly associated with tumor development and birth defects.