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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.


Pol κ depletion prevents the accumulation of DNA replication stress markers caused by p21 downmodulation.(A) Quantification of EdU positive cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in three independent experiments. (B) Quantification of CSK-resistant, PCNA positive U2OS cells transfected with the indicated siRNAs. 250 nuclei per sample were analyzed in three independent experiments. (C) Quantification of nuclei with more than 10 RPA foci in PCNA positive cells after transfection with the indicated siRNAs. 150 nuclei per sample were analyzed in three independent experiments. (D) Quantification of nuclear γH2AX intensity in cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.18020.014
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fig6s1: Pol κ depletion prevents the accumulation of DNA replication stress markers caused by p21 downmodulation.(A) Quantification of EdU positive cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in three independent experiments. (B) Quantification of CSK-resistant, PCNA positive U2OS cells transfected with the indicated siRNAs. 250 nuclei per sample were analyzed in three independent experiments. (C) Quantification of nuclei with more than 10 RPA foci in PCNA positive cells after transfection with the indicated siRNAs. 150 nuclei per sample were analyzed in three independent experiments. (D) Quantification of nuclear γH2AX intensity in cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.18020.014

Mentions: T. Huang and colleagues have previously reported that MN accumulation induced by USP1 depletion depends on Pol κ (Jones et al., 2012). Therefore, we set to explore the effect of p21 on Pol κ recruitment to DNA replication factories. First, we observed that Pol κ foci were formed only in a modest percentage of control cycling cells (siLuc in Figure 5A–B). However, when p21 was depleted, the percentage of cells with Pol κ foci raised significantly (Figure 5A–B). Second, the interaction of PCNA and GFP-Pol κ in the chromatin fraction increased when p21 was depleted (Figure 5C). Third, using PLA an increase in the number of endogenous PCNA/Pol κ interacting foci was revealed in p21-depleted samples (Figure 5D–E). We hypothesized that an increased recruitment of Pol κ to the replication forks in p21-depleted cell may slow down DNA elongation, triggering fork collapse and/or the generation of under-replicated DNA. To test this hypothesis, Pol κ was down-regulated in p21-depleted cells (Figure 6A) and different DNA replication parameters were tested. Forty eight hours after siRNA transfection, Pol κ depletion alone had no effect on most parameters, except from a modest increase in RPA foci formation (Figure 6—figure supplement 1). Such result may be in agreement with the role of Pol κ in the replication of non-B DNA regions such as G4 cuadruplex (Betous et al., 2009). Notably however, the simultaneous elimination of Pol κ and p21 prevented all the phenotypes associated with p21 depletion. Specifically, Pol κ depletion rescued the defective nascent DNA elongation (Figure 6B), the origin frequency (Figure 6C), the percentage of EdU positive cells (Figure 6—figure supplement 1A) and the increased number of cells with chromatin bound-PCNA (Figure 6—figure supplement 1B). Similarly, markers of replication stress such as RPA foci (Figure 6—figure supplement 1C), γH2AX (Figure 6—figure supplement 1D) and 53BP1 foci (Figure 6D) were downregulated after simultaneous depletion of p21 and Pol κ. Similar results were obtained when using a second siRNA specific for Pol κ in U2OS cells (Figure 6—figure supplement 2A–D) and when employing a different cell line, HCT116 p21 −/− cells (Figure 6—figure supplement 2E–F).10.7554/eLife.18020.012Figure 5.The recruitment of pol κ to replication-associated structures increases in the absence of p21.


Cyclin Kinase-independent role of p21 CDKN1A in the promotion of nascent DNA elongation in unstressed cells
Pol κ depletion prevents the accumulation of DNA replication stress markers caused by p21 downmodulation.(A) Quantification of EdU positive cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in three independent experiments. (B) Quantification of CSK-resistant, PCNA positive U2OS cells transfected with the indicated siRNAs. 250 nuclei per sample were analyzed in three independent experiments. (C) Quantification of nuclei with more than 10 RPA foci in PCNA positive cells after transfection with the indicated siRNAs. 150 nuclei per sample were analyzed in three independent experiments. (D) Quantification of nuclear γH2AX intensity in cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.18020.014
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fig6s1: Pol κ depletion prevents the accumulation of DNA replication stress markers caused by p21 downmodulation.(A) Quantification of EdU positive cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in three independent experiments. (B) Quantification of CSK-resistant, PCNA positive U2OS cells transfected with the indicated siRNAs. 250 nuclei per sample were analyzed in three independent experiments. (C) Quantification of nuclei with more than 10 RPA foci in PCNA positive cells after transfection with the indicated siRNAs. 150 nuclei per sample were analyzed in three independent experiments. (D) Quantification of nuclear γH2AX intensity in cells transfected with the indicated siRNAs. 200 nuclei per sample were analyzed in two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.18020.014
Mentions: T. Huang and colleagues have previously reported that MN accumulation induced by USP1 depletion depends on Pol κ (Jones et al., 2012). Therefore, we set to explore the effect of p21 on Pol κ recruitment to DNA replication factories. First, we observed that Pol κ foci were formed only in a modest percentage of control cycling cells (siLuc in Figure 5A–B). However, when p21 was depleted, the percentage of cells with Pol κ foci raised significantly (Figure 5A–B). Second, the interaction of PCNA and GFP-Pol κ in the chromatin fraction increased when p21 was depleted (Figure 5C). Third, using PLA an increase in the number of endogenous PCNA/Pol κ interacting foci was revealed in p21-depleted samples (Figure 5D–E). We hypothesized that an increased recruitment of Pol κ to the replication forks in p21-depleted cell may slow down DNA elongation, triggering fork collapse and/or the generation of under-replicated DNA. To test this hypothesis, Pol κ was down-regulated in p21-depleted cells (Figure 6A) and different DNA replication parameters were tested. Forty eight hours after siRNA transfection, Pol κ depletion alone had no effect on most parameters, except from a modest increase in RPA foci formation (Figure 6—figure supplement 1). Such result may be in agreement with the role of Pol κ in the replication of non-B DNA regions such as G4 cuadruplex (Betous et al., 2009). Notably however, the simultaneous elimination of Pol κ and p21 prevented all the phenotypes associated with p21 depletion. Specifically, Pol κ depletion rescued the defective nascent DNA elongation (Figure 6B), the origin frequency (Figure 6C), the percentage of EdU positive cells (Figure 6—figure supplement 1A) and the increased number of cells with chromatin bound-PCNA (Figure 6—figure supplement 1B). Similarly, markers of replication stress such as RPA foci (Figure 6—figure supplement 1C), γH2AX (Figure 6—figure supplement 1D) and 53BP1 foci (Figure 6D) were downregulated after simultaneous depletion of p21 and Pol κ. Similar results were obtained when using a second siRNA specific for Pol κ in U2OS cells (Figure 6—figure supplement 2A–D) and when employing a different cell line, HCT116 p21 −/− cells (Figure 6—figure supplement 2E–F).10.7554/eLife.18020.012Figure 5.The recruitment of pol κ to replication-associated structures increases in the absence of p21.

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.