Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

Mansilla, Sabrina F.; Bertolin, Agustina P.; Bergoglio, Valérie; Pillaire, Marie-Jeanne; Besteiro, Marina A. González; Luzzani, Carlos; Miriuka, Santiago Gabriel; Cazaux, Christophe; Hoffmann, Jean-Sébastien; Gottifredi, Vanesa

doi:10.7554/elife.18020

Cited by 32 publications

(52 citation statements)

References 70 publications

(121 reference statements)

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“…As such, we find that heterogeneity in G1 length is p53-dependent but p21-independent, while G1pm arrest is both p21- and p53-dependent. Moreover, that cells lacking p21 have increased levels of DNA damage suggests that p21 is important in maintaining long-term genome stability, consistent with recent work27.…”

Section: Resultssupporting

confidence: 88%

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

et al. 2017

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Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability.

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Section: Resultssupporting

confidence: 88%

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

et al. 2017

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“…On top of eliminating cells with damaged DNA by apoptosis or senescence, p53 is also capable of enhancing the processivity of DNA replication forks (6). Other groups reported similar findings, considering various mechanisms of how p53 might enhance DNA replication, e.g., through avoiding topological stress (7), inducing DNA polymerase-eta and translesion synthesis (8), orchestrating fork restart (9), or enhancing the levels of pCDKN1A/ p21 and its association with PCNA (10). In other systems, p53 can also compromise DNA replication through p53-associated exonuclease activity and DNA polymerase-iota (11), and forced CDKN1A/p21 synthesis impairs DNA replication in UV-irradiated cells (12).…”

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confidence: 78%

Chromatin modifiers Mdm2 and RNF2 prevent RNA:DNA hybrids that impair DNA replication

Ina

Wohlberedt

Magerhans

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The p53–Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.

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“…The shRNA insert (Peng et al, 2014) was cloned into the lentiviral transfer plasmid pLKO.1 (Addgene #8453) following manufacturer's instructions. Lentivirus production and infection was conducted exactly as previously described (Mansilla et al, 2016). When transfection was required, cells were infected 5 h after transfection.…”

Section: Sirnas and Expression Plasmidsmentioning

confidence: 99%

Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing

et al. 2019

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The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1‐inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK‐dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1‐deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1‐depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.

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Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells

Cited by 32 publications

References 70 publications

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

Chromatin modifiers Mdm2 and RNF2 prevent RNA:DNA hybrids that impair DNA replication

Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing

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