Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and genome instability that ensue when an origin is licensed on replicated DNA. However, the E3 ubiquitin ligase CRL4^Cdt2 only starts to degrade the licensing factor CDT1 after origin firing, raising the question of how cells prevent re-replication before CDT1 is fully degraded. Here, using quantitative microscopy and in-vitro-reconstituted human DNA replication, we show that CDT1 inhibits DNA synthesis during an overlap period when CDT1 is still present after origin firing. CDT1 inhibits DNA synthesis by suppressing CMG helicase at replication forks, and DNA synthesis commences once CDT1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis.

人类细胞在 G1 阶段许可数万个复制起点，然后必须在 S 期 DNA 合成之前停止所有许可，以防止在复制 DNA 上许可起点时发生的再复制和基因组不稳定。然而，E3 泛素连接酶 CRL4 ^Cdt2仅在原点激发后才开始降解许可因子 CDT1，这引发了细胞如何在 CDT1 完全降解之前防止再复制的问题。在这里，使用定量显微镜和体外-重构人类 DNA 复制，我们表明 CDT1 在重叠期间抑制 DNA 合成，此时 CDT1 在起源激发后仍然存在。CDT1 通过抑制复制叉处的 CMG 解旋酶来抑制 DNA 合成，一旦 CDT1 被降解，DNA 合成就会开始。因此，与人类细胞通过严格分离许可与发射、许可和发射重叠来防止再复制的流行模型相反，细胞反而将许可与 DNA 合成分开。