The events of the cell cycle are coordinated by feedback controls that ensure that cells do not enter mitosis unless their DNA has been replicated and do not leave mitosis unless a mitotic spindle has been correctly assembled. This proposal describes biochemical and genetic strategies to elucidate the molecular mechanism of cell cycle feedback control. The biochemical approach will use extracts of frog eggs which perform the cell cycle in vitro and lack detectable feedback controls. These extracts will be used as an assay system to purify and determine the mode of action of a prototypic feedback regulator, cytostatic factor (CSF), which arrests unfertilized frog eggs in meiosis be preventing the degradation of the cell cycle regulatory protein cyclin. CSF is inactivated at fertilization. The mechanism of CSF inactivation will also be investigated by examining the ability of somatic cell extracts to arrest the embryonic cell cycle has been arrested with inhibitors of DNA synthesis or spindle assembly will be tested for their ability to arrest the progress of in vitro cell cycle extracts. This complementation assay will be used to purify and characterize the components of cell cycle feedback control in somatic cells. Feedback control mutants that allow cells which have not completed DNA replication to enter mitosis will be isolated in budding yeast. These mutants will be characterized both genetically and biochemically. The study of cell cycle feedback controls is relevant to important medical problems. Failure of these controls leads to chromosome loss and breakage, causative events of some birth defects and cancers. Selective suppression of feedback controls in cancer cells could increase the power of chemotherapy.
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