Data for: Cdt1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis

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 CRL4Cdt2 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 progression 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.

Methods
Quantitative high-throughput microscopy was performed on human cell lines. Live-cell imaging of fluorescent cell cycle reporters in cell lines was quantified to study cell cycle dynamics, and this information was integrated together with fixed-cell imaging of immunofluorescene and other staining. Microscopy images were processed using a cell segmentation/tracking and quantification pipeline which automatically quantifies signals in single cells. The included data includes single-cell measurements for all experiments used to generate figures for Ratnayeke et al. 2022, as well as MATLAB scripts to generate the figures.

人类细胞在G1期会激活数万个复制起点，随后必须在S期DNA合成开始前终止所有复制许可过程，以防止复制起点在已复制的DNA上被激活后引发的再复制与基因组不稳定问题。然而，E3泛素连接酶（E3 ubiquitin ligase）CRL4Cdt2仅在复制起点启动后才开始降解复制许可因子Cdt1，这引出了一个关键科学问题：细胞如何在Cdt1完全降解前阻止再复制？本研究借助定量显微镜技术与体外重构的人类DNA复制体系，证实Cdt1会在复制起点启动后仍存在的重叠窗口期内抑制DNA合成。Cdt1通过抑制复制叉处的CMG解旋酶（CMG helicase）的推进来阻断DNA合成，而一旦Cdt1被降解，DNA合成即可启动。因此，与主流认为人类细胞通过严格分隔复制许可与起始过程来防止再复制的模型不同，人类细胞的复制许可与起始过程存在重叠，细胞实际是通过分隔复制许可与DNA合成来实现防再复制的。

实验方法
我们对人类细胞系开展了定量高通量显微镜成像。通过量化细胞系中荧光细胞周期报告基因的活细胞成像数据，研究细胞周期动态变化，并将该信息与免疫荧光及其他染色的固定细胞成像结果整合分析。显微镜图像通过细胞分割/追踪与定量流程进行处理，该流程可自动完成单细胞信号的量化分析。本数据集包含了生成Ratnayeke等人2022年论文图表所需的全部实验的单细胞测量数据，以及用于生成图表的MATLAB脚本。