Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Abstract

The authors find that human cells re-entering the cell cycle from quiescence have both an impaired p53-dependent DNA replication origin licensing checkpoint and slow origin licensing. This combination makes every first S phase underlicensed and hypersensitive to replication stress. ABSTRACTTo maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is Minichromosome Maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading prior to S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. As a result, the first S phase cells are hypersensitive to replication stress. This underlicensing is from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher risk cell cycle that promotes genome instability.

FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery

“…In this sense, various cell synchronization methods have been criticized for creating an artificial situation [ 10 ]. Indeed, recent evidence indicates that even a pause before initiating the cell cycle (i.e., entry to quiescence) will affect how the following S-phase is performed [ 11 ]. A prime example of a cell cycle pause is a DNA damage checkpoint.…”

Preparation for DNA replication: the key to a successful S phase

“…On the other hand, high p21 is also an indirect consequence of G1 arrest, since p21 is degraded in S phase cells and stable in G1 . We recently found that cells re‐entering the cell cycle from quiescence have an impaired licensing checkpoint . This natural checkpoint deficiency may provide a unique avenue to dissect the pathway connecting licensing to S phase entry.…”

“…When the checkpoint regulators are depleted or mutated, the cells can proceed prematurely into S phase, albeit with a higher risk of incomplete replication. Indeed, in nontransformed mammalian cell lines, manipulations that inhibit origin licensing result in a longer G1 phase and low CDK2 activity . Interestingly, the same manipulations in transformed cell lines do not affect G1 length or CDK2 activity, and cells instead proceed into S phase with considerably less than their typical amount of licensed origins.…”

“…Perhaps, the most compelling evidence for a true licensing checkpoint is the fact that removing p53, a central player in the response to cellular stresses that is absent or compromised in tumor‐derived cells, bypassed all of the checkpoint behaviors. Like transformed cells, nontransformed cells lacking p53 do not delay in G1 with low CDK2 activity even when licensing is impaired , and, like their yeast orthologs, can even enter premature M . Thus, it is clear that p53‐proficient cells can couple the activation of CDK2 and S phase with the status of origin licensing.…”

Μελέτη του παράγοντα αδειοδότησης της αντιγραφής Cdt1 καθώς και της υπεροικογένειας της Geminin στην πρόκληση γονιδιωματικής αστάθειας και στον καθορισμό της κυτταρικής μοίρας

“…Απορρύθμιση της αδειοδότησης της αντιγραφής είτε μέσω αποσιώπησης είτε μέσω υπερέκφρασης παραγόντων του προαντιγραφικού συμπλόκου θα μπορούσε να οδηγήσει σε αντιγραφικό στρες, γονιδιωματική αστάθεια και κυτταρικό θάνατο, ειδικότερα στα καρκινικά κύτταρα. Πιο συγκεκριμένα, αποσιώπηση της έκφρασης του CDT1 είτε υπερέκφραση της Geminin έχει δειχθεί ότι προκαλεί παύση του κυτταρικού κύκλου στην G1 φάση στα φυσιολογικά κύτταρα, ενώ ενεργοποιεί την διαδικασία της απόπτωσης στα καρκινικά κύτταρα (Shreeram et al, 2002;Nevis et al, 2009;Matson et al, 2017;Matson et al, 2019). Σε φυσιολογικά κύτταρα, έπειτα από περιορισμένη αδειοδότηση των αφετηριών της αντιγραφής, ενεργοποιείται το σημείο ελέγχου της αδειοδότησης, το οποίο προκαλεί παύση στη G1 φάση έως ότου ο κατάλληλος αριθμός των αφετηριών της αντιγραφής αδειοδοτηθεί (ΜcIntosh and Blow, 2012).…”