Consequences of partial chromosome re-replication in mammalian cells

  • Kathleen Klotz-Noack

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    To prevent re-replication of DNA in a single cell cycle, the licensing of
    replication origins by Mcm2-7 is prevented during S and G2 phases. Metazoans
    achieve this by cell cycle regulated proteolysis of the essential licensing factor
    Cdt1 and formation of an inhibitory heterohexameric complex of Cdt1 with a
    small protein called geminin. The consequences of either stabilising Cdt1 or
    ablating geminin in synchronised human U2OS cells are investigated in this
    PhD Thesis to elucidate the possible contribution of re-replication in gene
    amplifications or rearrangements commonly seen in human tumours. I show that following geminin loss, cells complete an apparently normal
    S-phase, but a proportion arrests at the G2/M boundary. When Cdt1 starts to
    accumulate in these cells, DNA re-replicates, suggesting that the key role of
    geminin is to prevent re-licensing in G2. Inhibition of cell cycle checkpoints in
    cells lacking geminin promotes progression through mitosis without detectable
    levels of re-replication. Checkpoint kinases thereby amplify re-replication into an
    all-or-nothing response by delaying geminin depleted cells in G2 phase.
    Comparative Genomic Hybridisation (CGH) array and Solexa Deep DNA
    sequencing revealed that re-replication after geminin depletion does not appear
    at preferential genomic regions within the human genome. This is consistent
    with a recent observation that G2 cells have lost their replication timing
    information and reduplicate their genome stochastically. In contrast, when Cdt1
    is stabilised by the neddylation inhibitor MLN4924, re-replication starts directly
    from within S-phase raising the question whether alternative mechanisms of may cause distinct genomic consequences.
    Date of Award2013
    Original languageEnglish
    SupervisorJulian Blow (Supervisor)

    Keywords

    • Chromosomes
    • Mammals
    • Cells

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