Ubiquitin-dependent proteolysis of mitotic cyclins and other proteins triggers sister chromosome segregation, cdc2 inactivation and exit from mitosis into G1 of the next cell cycle. The components responsible for this process include a novel ubiquitin carrier protein called E2-C and a 20S complex called the cyclosome (or APC, anaphase promoting complex). E2-C contains a highly conserved N-terminal extension that is, surprisingly, not needed for catalytic activity in vitro. A dominant negative E2-C (dnE2-C) blocks approximately 50 percent of cells in mitosis. As the only component of the cyclin destruction machinery for which a dominant negative currently exists, we will use dnE2-C in biochemical and genetic approaches to investigate the role of this machinery in mitosis and elsewhere in the cell cycle, and to identify its target proteins. We will also try to determine the role played by cdc20, a WD40 protein required for cyclin destruction and G2/M checkpoint control. This proposal is designed to answer the following questions about regulation of the cell cycle, using both biochemical and genetic approaches to study the cell cycles of both embryos and somatic cells. Information from these studies should help understand what molecular mechanisms control cell division in normal development and differentiation, and how defects in these mechanisms contribute to aneuploidy and the formation of cancer cells. Does E2-C function only in mitosis or, as recent evidence suggests, at other points in the cell cycle also? What is the function of the N- terminal extension? Does is act in a checkpoint pathway in G2 or mitosis? What is the role of cdc20 and its associated kinase during cell cycle progression leading to cyclin destruction? What turns off mitotic cyclin destruction at the end of G1 in mammalian cells? Why is the cyclin destruction machinery active in GO-arrested somatic cells?
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