Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Abstract

Abbreviations: APC/C, anaphase-promoting complex/cyclosome; ATM, Ataxia telangiectasia mutated kinase; ATR, Ataxia telangiectasia and Rad3 related kinase; AU, arbitrary units; Cdk, cyclin-dependent kinase; Chk1/2, checkpoint kinase 1/2; DDR, DNA damage response; DNA-PK, DNA-dependent protein kinase; LMB, Leptomycin B; Mdm2, mouse double minute 2 homolog; MK2, MAPKAP kinase 2; NCS, Neocarzinostatin; Plk1, polo-like kinase 1; gH2AX, histone variant; H2AX, phosphorylated on serine 139.Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C Cdh1 . However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C Cdh1 -mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle

“…While both G1 and G2 utilize all-or-none checkpoint kinetics, the G2 checkpoint is more stringent than G1 in the sense that the commitment point occurs very close (within 0.5 hr) to the G2/M border. This temporal location of the G2 commitment point is consistent with previous observations and may be concurrent with the onset of antephase described previously using time-lapse analysis (Xu et al , 2010; Krenning et al , 2014; Müllers et al , 2014, 2017; Feringa et al , 2016). In contrast, the G1 commitment point is located ~3–4 hrs before the G1/S border in both RPE and U2OS, meaning that DNA damage within the last 3 hrs of G1 usually fails to delay S phase onset.…”

Senescence from G2 arrest, revisited

“…93 Identical conclusions were reached in the recent paper by the Lindqvist team using a similar experimental approach. 96 In addition, by using fluorescent cyclin B1 and video-microscopy, they confirmed earlier observations 72 that p21-dependent sequestration of cyclin B1 does not take place in U2OS cells. In this p53/pRb proficient cell line, p21 induction is impaired due to inefficient ATM response 72 and, probably, mutation of the Wip1 phosphatase that interferes with p53 activation.…”

Hypersensitivity to DNA damage in antephase as a safeguard for genome stability

“…Excessive DNA damage in G2 cells can cause p21-dependent downregulation of Emi1 resulting in the APC/C Cdh1 activation and degradation of its targets1920. However, the latter response is limited to cells that contain high levels of damage, and follows only after p21-dependent nuclear retention of Cyclin B1–Cdk complexes79, not matching the fast response we observe in antephase cells. Therefore, we set out to test if Emi1 is needed to protect G2 cells from APC/C Cdh1 activation by DNA damage.…”