Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.

实现完整和精确的基因组复制需要每个基因组片段在每个细胞分裂周期中仅复制一次。保护大型真核基因组免于重新复制需要一组重叠的分子机制，以防止 S 期开始后的第一个 DNA 复制步骤，即 MCM 解旋酶复合物的 DNA 加载以许可复制起点。以前的报告已经定义了许多这样的起源许可抑制机制，但它们之间的时间关系尚不清楚，特别是在防止 G2 和 M 阶段的重复复制方面。结合人类细胞中的诱变、生物化学和单细胞分析，我们定义了一种新机制，通过必需的 MCM 加载蛋白 Cdt1 的过度磷酸化来防止再复制。我们证明了 Cyclin A/CDK1 可以使 Cdt1 过度磷酸化以抑制 G2 期 MCM 重新加载。抑制机制是阻断 Cdt1 与 MCM 的结合，独立于其他已知的 Cdt1 失活机制，例如 S 期期间的 Cdt1 降解或 Geminin 结合。此外，我们的研究结果表明，有丝分裂到 G1 相变中的 Cdt1 去磷酸化重新激活了 Cdt1。我们建议多个不同的、非冗余的许可抑制机制在每个细胞周期阶段以一系列顺序中继起作用，以确保精确的基因组复制。我们的研究结果表明，有丝分裂到 G1 相变中的 Cdt1 去磷酸化重新激活了 Cdt1。我们建议多个不同的、非冗余的许可抑制机制在每个细胞周期阶段以一系列顺序中继起作用，以确保精确的基因组复制。我们的研究结果表明，有丝分裂到 G1 相变中的 Cdt1 去磷酸化重新激活了 Cdt1。我们建议多个不同的、非冗余的许可抑制机制在每个细胞周期阶段以一系列顺序中继起作用，以确保精确的基因组复制。