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

Citation

Ratnayeke, Nalin; Chung, Mingyu; Meyer, Tobias (2021), Cdt1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis, Dryad, Dataset, https://doi.org/10.5061/dryad.4xgxd2599

Abstract

A fundamental concept in eukaryotic DNA replication is the temporal separation of G1 origin licensing from S phase origin firing. Re-replication and genome instability ensue if licensing occurs after DNA synthesis has started. In humans and other vertebrates, the E3 ubiquitin ligase CRL4Cdt2 starts to degrade the licensing factor Cdt1 after origins fire, raising the question of how cells prevent re-replication in early S phase. Here, using quantitative microscopy, we show that Cdt1 inhibits DNA synthesis during an overlap period when cells fire origins while Cdt1 is still present. Cdt1 inhibits DNA synthesis by suppressing CMG helicase progression at replication forks through the MCM-binding domain of Cdt1, and DNA synthesis commences once Cdt1 is degraded. Thus, instead of separating licensing from firing to prevent re-replication in early S phase, cells separate licensing from DNA synthesis through Cdt1-mediated inhibition of CMG helicase after firing.

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. 2021, as well as MATLAB scripts to generate the figures. 

Usage Notes

Code repository to generate figures can be found at DOI: 10.5281/zenodo.5037903 or https://github.com/MeyerLab/image-analysis-ratnayeke-2021. 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.