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 note that licensing checkpoint activity is only readily detected in cells with particularly slow MCM loading. In previous studies, MCM loading was artificially slowed to test for checkpoint proficiency in various genetic backgrounds (Shreeram et al., 2002; Liu et al., 2009; Nevis et al., 2009) . Under normal growth conditions however both checkpoint-proficient and checkpoint-impaired cell lines enter S phase with normal high levels of MCM loading. This behavior suggests that MCM loading is usually fast enough to achieve high levels of loaded MCM in advance of the other rate-limiting molecular events necessary to trigger CDK2 activation and S phase entry. It is only slow-loading cells that are visibly affected by the checkpoint. In that sense, the origin licensing checkpoint resembles other cell cycle checkpoints that operate in all cell cycles in that " ...elimination of the checkpoint may have catastrophic or subtle consequences depending on the prevailing conditions." (Hartwell and Weinert, 1989) For example, the . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 contribution of the mitotic spindle assembly checkpoint is most obvious when attachments are severely compromised (Lara-Gonzalez et al., 2012) . The effect of the spindle assembly checkpoint on the rate of unperturbed mitotic progression is relatively minor except in individual cells that fail to form kinetochore-microtubule attachments on time (Meraldi et al., 2004) . Similarly, we did detect a modest increase in the number of underlicensed S phase cells in otherwise unperturbed proliferating p53 null cells compared to p53 WT cells (Fig. 4) . We infer that these individual cells loaded MCM slowly, but then could not effectively delay S phase entry.