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

The unique features of G0 and the first G1 phase suggests that origin licensing may be distinctly regulated during cell cycle re-entry. We entertained the notion that the increased G1 length may allow more time for MCM loading, causing a meaningful increase in DNA-loaded MCM when entering S phase compared to subsequent proliferating cell cycles. To test this idea, we used single cell flow cytometry to measure the amount of loaded MCM at S phase entry and live cell imaging to measure cell cycle timing to determine if the amount of loaded MCM differs between cell cycle re-entry and active proliferation. Surprisingly, we discovered instead that cells re-entering the cell cycle from G0 are in fact, routinely and significantly underlicensed rendering them hypersensitive to replication stress compared to proliferating cells. This finding is consistent with a recent report that the first S phase experiences higher spontaneous replication stress, though the source of that endogenous stress was not identified (Daigh et al., 2018) . We report here that MCM loading is slow in the first G1 phase and furthermore, that the first cell cycle has a severely compromised origin licensing checkpoint relative to the robust checkpoint in actively proliferating cells. This combination promotes premature S phase entry and underlicensing. To our knowledge, these results demonstrate the first naturally underlicensed cell cycle and suggest that We then applied this method to measure how much MCM had been loaded by the time of the G1/S transition in the first cell cycle compared to the amount loaded at G1/S in the second cell cycle (Fig. 1B) . We arrested RPE1-hTert cells in G0 by contact inhibition (in the presence of growth serum) for 48 hours. Contact-inhibited G0 cells showed a robust cell cycle exit to G0 with very little loaded MCM; 94% of cells were G0/G1 but only 1% were still in S phase ( Fig. 1C and Fig. S1B ). We then released cells to re-enter the cell cycle by plating at sub-confluent cell density. G0 cells also expressed the expected high levels of p27 and low Cyclin D1 compared to cells in the first cycle (Fig. S1C) . We pulse-labeled cells with EdU and harvested some cells in G0 or at 24 hours when they were a mix of first G1 and ~50-70% first S phase cells. In a separate continuous EdU labeling experiment in which EdU was added at the time of release, we determined that nearly 100% of cells had entered S phase by 28 hours (Fig. S1D) , and almost no cells remained in G0/G1. Thus, we concluded that additional cells we harvested 48 hours after release had completed their first S phase and were into their second cell cycle.