Investigating the Mechanism of Geminin Inactivation

  • Kevin Creavin

    Student thesis: Doctoral ThesisDoctor of Philosophy


    DNA replication is regulated throughout the cell cycle to ensure that the entire genome is replicated once per cell cycle. Replication licensing is a key process required for the initiation of DNA replication. Replication licensing refers to the chromatin loading of an essential replication factor, the MCM2-7 helicase. This is loaded onto DNA early in the cell cycle. MCM2-7 stay bound on chromatin until cells enter S-phase where CDKs act upon the MCM2-7 complex leading to the initiation of DNA replication. The processes of replication licensing and initiation are separated during the cell cycle to ensure these activities do not overlap as this can result in the rereplciation of DNA. Separation of the two processes enables licensing inhibition prior to the activation of CDKs. A key regulator of MCM loading is a coiled-coil protein called geminin. Geminin potently inhibits licensing by binding and inhibiting the replication licensing factor Cdt1. In somatic cell cycles geminin is degraded by the 26S proteasome to allow licensing to take place. However, in embryonic cell cycles geminin remains stable throughout the cell cycle. This is essential as geminin is a duel function protein. It is required in embryonic cell cycles to maintain pluripotency; therefore it must remain stable in these cell types. It is also a potent inhibitor of DNA replication; therefore it must be subjected to some form of inactivation.
    The aim of this project was to identify the molecular mechanism of geminin inactivation in Xenopus egg extracts. Experiments undertaken during this project have demonstrated that geminin is both stable and inactive in Xenopus egg extracts. In addition some factors essential for the inactivation of geminin have been identified. The kinetics of inactivation have been determined and is has been demonstrated that interphase inactive geminin is biochemically distinct from active metaphase geminin.
    Date of Award2015
    Original languageEnglish
    SupervisorJulian Blow (Supervisor)

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