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Cyclin Kinase-independent role of p21 CDKN1A in the promotion of nascent DNA elongation in unstressed cells

View Article: PubMed Central - PubMed

ABSTRACT

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

Doi:: http://dx.doi.org/10.7554/eLife.18020.001

No MeSH data available.


Related in: MedlinePlus

The PIR domain is required for p21 recruitment to replication factoriesU2OS cells were transfected with GFP-PCNA and p21 or p21PIPMut* respectively.(A and C) Representative images of panuclear and foci distribution of GFP-PCNA and p21 is shown for each non-extracted condition. (B and D) After pre-extraction with CSK buffer, the colocalization of p21 and PCNA was analyzed by confocal microscopy, generating profiles of signal intensity along an arbitrary line drawn across the nuclei. (E) p21 and p21PIPMut*expressing cells were subjected to CSK extraction for the indicated times. (F) The p21 relative retention into the insoluble fraction was quantified by densitometric analysis. The values on the plot were normalized to the KU70 intensity for each sample.DOI:http://dx.doi.org/10.7554/eLife.18020.006
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fig1s3: The PIR domain is required for p21 recruitment to replication factoriesU2OS cells were transfected with GFP-PCNA and p21 or p21PIPMut* respectively.(A and C) Representative images of panuclear and foci distribution of GFP-PCNA and p21 is shown for each non-extracted condition. (B and D) After pre-extraction with CSK buffer, the colocalization of p21 and PCNA was analyzed by confocal microscopy, generating profiles of signal intensity along an arbitrary line drawn across the nuclei. (E) p21 and p21PIPMut*expressing cells were subjected to CSK extraction for the indicated times. (F) The p21 relative retention into the insoluble fraction was quantified by densitometric analysis. The values on the plot were normalized to the KU70 intensity for each sample.DOI:http://dx.doi.org/10.7554/eLife.18020.006

Mentions: The limited amounts of p21 in cycling cells allow CDK-dependent cell cycle progression (Kreis et al., 2014). Indeed, p21 levels in cycling cells are not and can be detected on EdU positive cells with p21 specific antibodies (Figure 1A and B) as reported recently (Coleman et al., 2015). Remarkably, while p21 levels are at the lowest in S phase (Figure 1—figure supplement 1A,B), they are sufficient to impair TLS-dependent DNA synthesis (Mansilla et al., 2013; Soria and Gottifredi, 2010) if not degraded after UV irradiation (Figure 1—figure supplement 1A, B). Notably, the function of p21 during unperturbed cell cycle progression remained unknown. A hint of such function was revealed by a Proximity ligation assay (PLA) which revealed a chromatin bound PCNA/p21 interaction in cycling cells. Such complexes resisted a mild extraction with CSK buffer which removes proteins unbound to chromatin (Figure 1C–D). Consistent with our previous findings, the percentage of cells with PLA spots was reduced by UV irradiation and PLA spots were not detected upon p21 depletion (Figure 1C–D). In agreement, endogenous p21 colocalized with PCNA (Figure 1E and F) and EdU-labelled replication factories (Figure 1—figure supplement 1C). The colocalization of p21 and GFP-PCNA became more evident following removal of proteins unbound to chromatin (Figure 1—figure supplement 2A). We next evaluated the requirement of the p21 PIR region for p21-PCNA colocalization. To this end, we transfected cells with either p21 or p21PIPMut*, bearing an intact or a disrupted PIR, respectively (Mansilla et al., 2013; Soria et al., 2008). The disruption of the CDK-binding site by point mutations in both constructs (Mansilla et al., 2013; Soria et al., 2006, 2008), prevented the arrest outside S phase expected after p21 overexpression (Figure 1—figure supplement 2B,C). Similar to endogenous p21, overexpressed p21 localized to replication factories (Figure 1G and Figure 1—figure supplement 3A,B). However, p21PIPMut* lost its ability to form foci at replication factories (Figure 1H and Figure 1—figure supplement 3C), did not colocalize with PCNA (Figure 1—figure supplement 3C) and showed reduced chromatin retention (Figure 1—figure supplement 3D and E). Hence, the PIR of p21 is required for the localization of p21 to replication factories in cycling cells.10.7554/eLife.18020.003Figure 1.The PCNA interacting region of p21 facilitates the recruitment of p21 to replication factories in cycling cells.


Cyclin Kinase-independent role of p21 CDKN1A in the promotion of nascent DNA elongation in unstressed cells
The PIR domain is required for p21 recruitment to replication factoriesU2OS cells were transfected with GFP-PCNA and p21 or p21PIPMut* respectively.(A and C) Representative images of panuclear and foci distribution of GFP-PCNA and p21 is shown for each non-extracted condition. (B and D) After pre-extraction with CSK buffer, the colocalization of p21 and PCNA was analyzed by confocal microscopy, generating profiles of signal intensity along an arbitrary line drawn across the nuclei. (E) p21 and p21PIPMut*expressing cells were subjected to CSK extraction for the indicated times. (F) The p21 relative retention into the insoluble fraction was quantified by densitometric analysis. The values on the plot were normalized to the KU70 intensity for each sample.DOI:http://dx.doi.org/10.7554/eLife.18020.006
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Related In: Results  -  Collection

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fig1s3: The PIR domain is required for p21 recruitment to replication factoriesU2OS cells were transfected with GFP-PCNA and p21 or p21PIPMut* respectively.(A and C) Representative images of panuclear and foci distribution of GFP-PCNA and p21 is shown for each non-extracted condition. (B and D) After pre-extraction with CSK buffer, the colocalization of p21 and PCNA was analyzed by confocal microscopy, generating profiles of signal intensity along an arbitrary line drawn across the nuclei. (E) p21 and p21PIPMut*expressing cells were subjected to CSK extraction for the indicated times. (F) The p21 relative retention into the insoluble fraction was quantified by densitometric analysis. The values on the plot were normalized to the KU70 intensity for each sample.DOI:http://dx.doi.org/10.7554/eLife.18020.006
Mentions: The limited amounts of p21 in cycling cells allow CDK-dependent cell cycle progression (Kreis et al., 2014). Indeed, p21 levels in cycling cells are not and can be detected on EdU positive cells with p21 specific antibodies (Figure 1A and B) as reported recently (Coleman et al., 2015). Remarkably, while p21 levels are at the lowest in S phase (Figure 1—figure supplement 1A,B), they are sufficient to impair TLS-dependent DNA synthesis (Mansilla et al., 2013; Soria and Gottifredi, 2010) if not degraded after UV irradiation (Figure 1—figure supplement 1A, B). Notably, the function of p21 during unperturbed cell cycle progression remained unknown. A hint of such function was revealed by a Proximity ligation assay (PLA) which revealed a chromatin bound PCNA/p21 interaction in cycling cells. Such complexes resisted a mild extraction with CSK buffer which removes proteins unbound to chromatin (Figure 1C–D). Consistent with our previous findings, the percentage of cells with PLA spots was reduced by UV irradiation and PLA spots were not detected upon p21 depletion (Figure 1C–D). In agreement, endogenous p21 colocalized with PCNA (Figure 1E and F) and EdU-labelled replication factories (Figure 1—figure supplement 1C). The colocalization of p21 and GFP-PCNA became more evident following removal of proteins unbound to chromatin (Figure 1—figure supplement 2A). We next evaluated the requirement of the p21 PIR region for p21-PCNA colocalization. To this end, we transfected cells with either p21 or p21PIPMut*, bearing an intact or a disrupted PIR, respectively (Mansilla et al., 2013; Soria et al., 2008). The disruption of the CDK-binding site by point mutations in both constructs (Mansilla et al., 2013; Soria et al., 2006, 2008), prevented the arrest outside S phase expected after p21 overexpression (Figure 1—figure supplement 2B,C). Similar to endogenous p21, overexpressed p21 localized to replication factories (Figure 1G and Figure 1—figure supplement 3A,B). However, p21PIPMut* lost its ability to form foci at replication factories (Figure 1H and Figure 1—figure supplement 3C), did not colocalize with PCNA (Figure 1—figure supplement 3C) and showed reduced chromatin retention (Figure 1—figure supplement 3D and E). Hence, the PIR of p21 is required for the localization of p21 to replication factories in cycling cells.10.7554/eLife.18020.003Figure 1.The PCNA interacting region of p21 facilitates the recruitment of p21 to replication factories in cycling cells.

View Article: PubMed Central - PubMed

ABSTRACT

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

Doi:: http://dx.doi.org/10.7554/eLife.18020.001

No MeSH data available.


Related in: MedlinePlus