Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, UK.
J Cell Sci. 2012 May 15;125(Pt 10):2436-45. doi: 10.1242/jcs.100883. Epub 2012 Feb 24.
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.
为了防止在单个细胞周期中 DNA 的重复复制，Mcm2-7 在 S 和 G2 期阻止复制原点的许可。动物细胞通过细胞周期调控的必需许可因子 Cdt1 的蛋白水解和 geminin 对 Cdt1 的抑制来实现这一点。在这里，我们研究了在同步化的人 U2OS 细胞中敲除 geminin 的后果。在 geminin 缺失后，细胞完成了一个明显正常的 S 期，但一部分细胞在 G2-M 边界处停滞。当这些细胞中 Cdt1 积累时，DNA 会重新复制，这表明 geminin 的关键作用是防止 G2 中的重新许可。如果细胞周期检查点在缺乏 geminin 的细胞中被抑制，细胞就会通过有丝分裂，发生的再复制就会减少。检查点激酶通过延迟 G2 期缺乏 geminin 的细胞来放大再复制，从而形成全有或全无的反应。深度 DNA 测序显示，在 geminin 耗尽后，没有特定基因组区域的优先再复制。这与这样的观察结果一致，即在 G2 期的细胞已经失去了它们的复制时间信息。相比之下，当 Cdt1 过表达或被 neddylation 抑制剂 MLN4924 稳定时，再复制可以发生在整个 S 期。
