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

Full origin licensing checkpoint activity in normal human fibroblasts requires the p53 tumor suppressor (Fig. 4A ) . To test if p53-deficient epithelial cells are more likely to enter S phase underlicensed, we compared isogenic WT and p53 homozygous null RPE1-hTert cells (gift of P. Jallepalli, Rodriguez-Rodriguez et al., 2018) . We analyzed the effects of reduced origin licensing from Cdt1 depletion on both G1 length and early S phase licensing status. We first noted by immunoblotting that Cdt1 depletion induced accumulation of both p53 and the CDK2 inhibitor p21, the product of a p53-inducible gene, whereas p53 null cells lacked both p53 and detectable levels of p21 (Fig. 4B ). The absence of p53 had little effect on either G1 phase MCM loading (Fig. S3C ) or the amount of MCM loaded by early S phase in otherwise unperturbed cells (compare black and grey lines in Fig 4C, fold change in Fig. 4D , and percentage of underlicensed cells in Fig 4E) . As before, Cdt1 depletion slowed MCM loading in WT cells and increased G1 length by several-fold (Fig. 4F, Fig. S3D ). In contrast, Cdt1-depleted cells lacking p53 entered S phase with significantly less MCM loaded (compare red and orange lines in Fig. 4C , fold change in Fig. 4D , and . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/558783 doi: bioRxiv preprint percentage of underlicensed cells in Fig. 4E ). Moreover, unlike p53 WT cells, Cdt1 depletion in p53 null cells did not cause G1 lengthening (Fig. 4F) . Thus, loss of p53 cripples the origin licensing checkpoint in proliferating cells, allowing inappropriate premature S phase entry of underlicensed cells. We note that even in control cells that had not been depleted of Cdt1, the p53 null cells entered S phase at a slightly lower amount of loaded MCM on average compared to p53 WT cells (compare black and red lines in Fig. 4C) . We also detected a modest increase in the percentage of underlicensed p53 null siControl cells (Fig. 4E ). Overall we conclude that these untransformed epithelial cells utilize a p53-dependent checkpoint to couple the timing of S phase entry to the status of origin licensing.