Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation

Kathleen Klotz-Noack, Debbie McIntosh, Nicholas Schurch, Norman Pratt, J. Julian Blow

    Research output: Contribution to journalArticle

    37 Citations (Scopus)

    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. Animal cells achieve this by cell-cycle-regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2-M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. By contrast, when Cdt1 is overexpressed or is stabilised by the neddylation inhibitor MLN4924, re-replication can occur throughout S phase.

    Original languageEnglish
    Pages (from-to)2436-2445
    Number of pages10
    JournalJournal of Cell Science
    Volume125
    Issue number10
    DOIs
    Publication statusPublished - 15 May 2012

    Cite this

    Klotz-Noack, Kathleen ; McIntosh, Debbie ; Schurch, Nicholas ; Pratt, Norman ; Blow, J. Julian. / Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation. In: Journal of Cell Science. 2012 ; Vol. 125, No. 10. pp. 2436-2445.
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    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. Animal cells achieve this by cell-cycle-regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2-M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. By contrast, when Cdt1 is overexpressed or is stabilised by the neddylation inhibitor MLN4924, re-replication can occur throughout S phase.",
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    Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation. / Klotz-Noack, Kathleen; McIntosh, Debbie; Schurch, Nicholas; Pratt, Norman; Blow, J. Julian.

    In: Journal of Cell Science, Vol. 125, No. 10, 15.05.2012, p. 2436-2445.

    Research output: Contribution to journalArticle

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