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 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.

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. 

Code repository to generate figures can be found at https://doi.org/10.5281/zenodo.7183751 or https://github.com/MeyerLab/image-analysis-ratnayeke-2022. See README in code repository for instructions. Figure generating scripts are directly executable once the path to the data folder in each script is edited.