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

Origin licensing checkpoint activity is cell type-dependent however (Shreeram et al., 2002) . We therefore sought first to assess checkpoint status in proliferating untransformed epithelial cells (RPE1-hTert). We decreased origin licensing with siRNA targeting Cdc10 dependent transcript 1 (Cdt1), an essential MCM loading protein (Pozo and Cook, 2016) . We used either a pool of four siCdt1 sequences (siCdt1 A) or a single independent siCdt1 (siCdt1 B) and analyzed Cdt1 protein by immunoblotting (Fig. 3B) . We then tested both MCM loading and the length of G1 phase. Cdt1 depletion induced both a reduction in the rate of G1 phase MCM loading (Fig. S3A, B) and also a striking G1 lengthening. G1 length dramatically increased by several-fold (12 to 42 hours for siCdt1B), and the more profound Cdt1 depletion by siCdt1 B caused a greater G1 delay (Fig. 3F , green bars). Importantly, Cdt1 depletion alone changed neither the amount of early S loaded MCM (compare black and grey lines in Fig. 3C , and fold change in Fig. 3D ) nor the percentage of underlicensed early S phase cells (Fig. 3E ). By the time cells finally entered S phase, they had achieved normal amounts of loaded MCM. These observations indicate that actively proliferating RPE1 cells wait for the normal amount of loaded MCM in G1 phase before entering S phase.