Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Jacob Peter Matson; Amy M. House; Gavin D. Grant; Huaitong Wu; Joanna Perez; Jeanette Gowen Cook

Many questions remain about the origin licensing checkpoint itself. How do cells monitor the status of origin licensing, what conditions satisfy the checkpoint, and how is licensing status coupled to the activation of CDKs? Our results indicate that a 50% reduction in the amount of loaded MCM by the time of S phase entry sensitizes human cells to exogenous replication stress; this level is similar to that reported for checkpointdeficient U2OS cells (Ge et al., 2007) . Some stem cell types have a greater amount of loaded MCM at S phase entry than their differentiated counterparts, suggesting that the licensing threshold may vary by cell type (Ge et al., 2015; Carroll et al., 2018) . The mere existence of the licensing checkpoint was an unresolved question until relatively recently in large part because it appears to be restricted to untransformed metazoan cells (Piatti et al., 1995) . This restriction poses technical challenges for experimentally interrogating the checkpoint because untransformed cells are more difficult to synchronize and manipulate than tumor-derived cells or yeast cells. The fact that p53 status correlates with licensing checkpoint proficiency gives clues that a p53-dependent process, perhaps one or more p53-regulated genes, is involved in efficiently coupling licensing to S phase entry. The discovery here that cell cycle re-entry induces natural underlicensing also suggests future avenues to identify checkpoint mediators. Our analysis of origin licensing status at the G1/S transition using analytical flow cytometry provides a promising opportunity to dissect this pathway in the future.