title: Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation.
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.
authors.affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Klotz-Noack
authors.forename: Debbie
authors.initials: D
authors.lastname: McIntosh
authors.forename: Nicholas
authors.initials: N
authors.lastname: Schurch
authors.forename: Norman
authors.initials: N
authors.lastname: Pratt
authors.forename: J Julian
authors.initials: JJ
authors.lastname: Blow
mesh_terms: D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002477:Cells / Q000166:cytology* / Q000378:metabolism; D004261:DNA Replication; D016195:G2 Phase; D064248:Geminin; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase
chemical_list: C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; C577995:GMNN protein, human; D064248:Geminin; D009687:Nuclear Proteins; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2