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

A cell cycle checkpoint that delays progression to the next phase is distinct from a simple incapacity to proceed to the next phase because it is possible for genetic alterations to bypass a checkpoint and induce premature cell cycle progression (Hartwell and Weinert, 1989) . The origin licensing checkpoint delays the activation of Cyclin E/CDK2 (Fig. 3A ) . To test if the G1 phase delay can be bypassed in Cdt1-depleted RPE1-hTert cells, we overproduced Cyclin E1 to prematurely activate CDK2. We used flow cytometry to measure DNA-loaded MCM, plotting a histogram of early S phase loaded MCM as before ( Fig. 3C and Fig. S3A ). As expected from prior reports, Cyclin E1 overproduction in control cells shortened G1 phase nearly three-fold (siControl vs ↑Cyclin E1, Fig. 3F , green bars) (Matson et al., 2017; Resnitzky et al., 1994) . In addition Cyclin E1 overproduction alone induced underlicensing as measured by the amount of early S loaded MCM (compare black and red lines, Fig. 3C , and the percentage of underlicensed cells, Fig. 3E ) (Ekholm-Reed et al., 2004; Matson et al., 2017) . Strikingly, when we overproduced Cyclin E1 after Cdt1 depletion (Fig. 3A) , early S phase cells became severely underlicensed, quantified by both MCM loaded per cell (compare grey and orange lines, Fig. 3C and fold change in Fig. 3D ), and the percentage of underlicensed cells (Fig. 3E) . Moreover, G1 length decreased even in the Cdt1-depleted cells (Fig. 3F) . We previously determined that the apparent decease in Cdt1 protein upon Cyclin E1 overproduction is an indirect effect of cell cycle phase distribution because Cdt1 is stable in G1 and unstable in S phase (Matson et al., 2017) . Since Cyclin E1 overproduction bypassed the strong G1 delay in control Cdt1-depleted cells and induced S phase entry with low amounts of loaded MCM, the G1 delay is caused by a bona fide origin licensing checkpoint.