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

We had established in Figures 1 and 2 that cells re-entering the first cell cycle after G0 are routinely underlicensed relative to subsequent cycles. We hypothesized that the first cell cycle has an impaired origin licensing checkpoint that poorly couples the length of G1 phase to the status of MCM loading. To test that hypothesis, we compared actively proliferating cells treated with siCdt1 to G0 cells re-entering the first cycle also treated with siCdt1 (Fig. 5A ). We measured cell cycle phase distribution by DNA Content (DAPI) and DNA synthesis (EdU) (Fig. 5B ). The actively proliferating cells treated with siCdt1 increased the percentage of G1 cells as before, but cells re-entering the first cell cycle from G0 did not similarly respond to Cdt1 depletion (Fig. 5B , green bars). This difference is consistent with a weak origin licensing checkpoint in the first cell cycle. We also tested if the loss of p53 in the first cell cycle enhanced this apparent checkpoint deficiency (Fig. 5C ). Unlike the proliferating cells in Fig. 4F , first cell cycle p53 null cells were no worse than first cell cycle WT cells (Fig. 5D ). We note that siControl p53 null cells re-entering the first cycle also started S phase sooner than their corresponding p53 WT cells based on cell cycle distributions at the same time after G0 release (Fig. 5B 33% G1 vs, 16 .6% G1 in Fig. 5D ). The faster S phase entry by the p53 null cells could be due to both the impaired licensing checkpoint and the general loss of basal p21 protein ( Fig. 5C ) (Overton et al., 2014) , among other possible p53-dependent effects on G1/S progression. The ability of actively proliferating cells to activate the licensing checkpoint to extend G1 combined with the observation that G0 cells do not extend G1 but instead enter S phase underlicensed strongly suggests that cells in the first cell cycle after G0 have an impaired origin licensing checkpoint. more slowly than cells in the second cycle. In both cases, the compromised origin licensing checkpoint is unable to extend G1 in response to the low MCM loading status resulting in an underlicensed first S phase. To distinguish between those explanations, we measured the nuclear accumulation of the Cell Division Cycle 6 (Cdc6) protein, an essential MCM loading protein. Cdc6 is degraded by the Anaphase promoting complex-Cdh1 (APC Cdh1 ) in G1 phase, both in cells re-entering the cell cycle and in proliferating cells (Petersen et al., 2000; Mailand and Diffley, 2005) . CDK2/Cyclin E1 phosphorylates Cdc6 in late G1 to protect it from APC Cdh1 , allowing Cdc6 protein to accumulate (Mailand and Diffley, 2005) . Because Cdc6 is essential for MCM loading, Cdc6 accumulation is one of the limiting steps for MCM loading in G1. The time for MCM loading ends when S phase starts and Cdc6 is exported from the nucleus to the cytoplasm (Petersen et al., 1999) in addition to many other mechanisms that inhibit rereplication (Truong and Wu, 2011) . Therefore, the time Cdc6 is detectable in nuclei is one proxy for the length of maximum available MCM loading time in G1 phase. Nuclear Cdc6 is also a marker for the functional border between G0/early G1 when MCM is not loaded (or loaded very slowly) and late G1 when MCM can be loaded.