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

Cells re-entering the cell cycle from G0 license origins slowly and begin S phase before they are fully licensed. We hypothesized that artificially extending the first G1 phase could rescue the underlicensing of the first S phase by extending time for MCM loading. If successful, then cells would enter S phase with the high amount of loaded MCM typical of proliferating cells. We chose to extend G1 phase using nutlin-3a, a p53 stabilizing drug previously shown to lengthen G1 (Tovar et al., 2006) . We treated cells re-entering the first G1 phase with nutlin-3a beginning in mid-G1 for 8 hours, then washed off the drug to permit passage into S phase and measured the amount of loaded MCM at S phase entry (Fig. 7A, B) . Nutlin-3a stabilized p53, causing p21 protein accumulation. This effect is known to inhibit CDK2 kinase activity and delay S phase entry (Giono and Manfredi, 2007) . An unwanted secondary effect of low CDK2 activity for our purposes is failure to protect Cdc6 in late G1 phase, which would interfere with our goal of extending the time for MCM loading (Petersen et al., 2000; Mailand and Diffley, 2005) . To allow MCM loading during the nutlin-induced G1 arrest, we . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 constitutively expressed a mutant form of Cdc6 that does not require CDK2 activity for stability (Matson et al., 2017) . We then compared licensing and cell cycle progression in these treated cells to untreated cells from the first and second cell cycles (Fig. 7C, Fig. S4A ). Strikingly, transiently extending the first G1 phase by several hours almost fully rescued licensing in the first cell cycle to the same high level as in the second cell cycle (compare green and grey lines, Fig. 7C , and fold change Fig. 7D ) and decreased the percentage of underlicensed cells to the same low level as the second cell cycle (Fig. 7E ). Unlike the effects of stable Cdc6 on the rate of MCM loading in proliferating cells (Matson et al., 2017) , neither stable Cdc6 alone nor in combination with Cdt1 overproduction increased the rate of MCM loading in the first G1 phase ( Fig. S4B and S4C). These genetic changes also had little-to-no effect on underlicensing in the first S phase ( Fig. S4D-F) . Therefore, the improved licensing by early S phase from nutlininduced G1 extension is due to the increased time for MCM loading, rather than directly improving MCM loading itself.