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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.


Mechanistically distinct regulators of alt DNA pols are required to facilitate unperturbed DNA replication.(A) W.B. analysis revealing PCNA, ubi-PCNA and p21 levels in U2OS cells transfected with the indicated siRNAs. (B) USP1 mRNA levels were examined by quantitative RT-PCR. (C) Model depicting the different mechanisms of PCNA regulation by p21 and USP1. (D) IdU track length was measured in 85 fibers/sample in three independent experiments. (E) Origin firing frequency. 150 fibers/sample were analysed in three independent experiments. (F) Quantification of cells with more than five 53BP1 foci. 200 U2OS cells/sample were analysed in three independent experiments. (G) MN accumulation. 300 binucleated cells/sample were analyzed in three independent experiments. Data on the effect of USP1 downregulation on the modulation of nascent DNA elongation (Figure 4D) and accumulation of cells with 53BP1 foci (Figure 4F) and micronuclei (Figure 4G) was reproduced from Jones et al, EMBO.DOI:http://dx.doi.org/10.7554/eLife.18020.011
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fig4: Mechanistically distinct regulators of alt DNA pols are required to facilitate unperturbed DNA replication.(A) W.B. analysis revealing PCNA, ubi-PCNA and p21 levels in U2OS cells transfected with the indicated siRNAs. (B) USP1 mRNA levels were examined by quantitative RT-PCR. (C) Model depicting the different mechanisms of PCNA regulation by p21 and USP1. (D) IdU track length was measured in 85 fibers/sample in three independent experiments. (E) Origin firing frequency. 150 fibers/sample were analysed in three independent experiments. (F) Quantification of cells with more than five 53BP1 foci. 200 U2OS cells/sample were analysed in three independent experiments. (G) MN accumulation. 300 binucleated cells/sample were analyzed in three independent experiments. Data on the effect of USP1 downregulation on the modulation of nascent DNA elongation (Figure 4D) and accumulation of cells with 53BP1 foci (Figure 4F) and micronuclei (Figure 4G) was reproduced from Jones et al, EMBO.DOI:http://dx.doi.org/10.7554/eLife.18020.011

Mentions: Besides p21, novel negative regulators of alt DNA pols have been recently identified (Bertolin et al., 2015). One of them, USP1, has the ability to remove the ubiquitin moiety from PCNA (Huang et al., 2006; Niimi et al., 2008). Mono-ubiquitinated PCNA interacts with the UBM and UBZ domains of alternative DNA pols favouring their loading to the replisomes (Bienko et al., 2005; Kannouche and Lehmann, 2004; Plosky et al., 2006). During unperturbed replication, PCNA ubiquitination is limited by USP1 (Huang et al., 2006; Niimi et al., 2008), as evidenced by increased PCNA ubiquitination upon USP1 depletion (Figure 4A and B). In constrast, the level of PCNA ubiquitination was not modified when p21 was depleted (Figure 4A). Hence, p21 and USP1 regulate the recruitment of alt pols by distinct mechanisms (Figure 4C). Intriguingly, despite such mechanistic differences, both p21 and USP1 depletion caused a similar effect (both in quality and extent) on nascent DNA elongation (Figure 4D), origin frequency (Figure 4E), the accumulation of cells with 53BP1 foci (Figure 4F) and the number of binucleated cells with MN (Figure 4G). These results reinforce the function of p21 as a negative regulator of alt DNA pols. Moreover, mechanistically distinct regulators of alt DNA pols are equally required to preserve DNA replication and genomic stability during unperturbed replication.10.7554/eLife.18020.011Figure 4.Mechanistically distinct regulators of alt DNA pols are required to facilitate unperturbed DNA replication.


Cyclin Kinase-independent role of p21 CDKN1A in the promotion of nascent DNA elongation in unstressed cells
Mechanistically distinct regulators of alt DNA pols are required to facilitate unperturbed DNA replication.(A) W.B. analysis revealing PCNA, ubi-PCNA and p21 levels in U2OS cells transfected with the indicated siRNAs. (B) USP1 mRNA levels were examined by quantitative RT-PCR. (C) Model depicting the different mechanisms of PCNA regulation by p21 and USP1. (D) IdU track length was measured in 85 fibers/sample in three independent experiments. (E) Origin firing frequency. 150 fibers/sample were analysed in three independent experiments. (F) Quantification of cells with more than five 53BP1 foci. 200 U2OS cells/sample were analysed in three independent experiments. (G) MN accumulation. 300 binucleated cells/sample were analyzed in three independent experiments. Data on the effect of USP1 downregulation on the modulation of nascent DNA elongation (Figure 4D) and accumulation of cells with 53BP1 foci (Figure 4F) and micronuclei (Figure 4G) was reproduced from Jones et al, EMBO.DOI:http://dx.doi.org/10.7554/eLife.18020.011
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fig4: Mechanistically distinct regulators of alt DNA pols are required to facilitate unperturbed DNA replication.(A) W.B. analysis revealing PCNA, ubi-PCNA and p21 levels in U2OS cells transfected with the indicated siRNAs. (B) USP1 mRNA levels were examined by quantitative RT-PCR. (C) Model depicting the different mechanisms of PCNA regulation by p21 and USP1. (D) IdU track length was measured in 85 fibers/sample in three independent experiments. (E) Origin firing frequency. 150 fibers/sample were analysed in three independent experiments. (F) Quantification of cells with more than five 53BP1 foci. 200 U2OS cells/sample were analysed in three independent experiments. (G) MN accumulation. 300 binucleated cells/sample were analyzed in three independent experiments. Data on the effect of USP1 downregulation on the modulation of nascent DNA elongation (Figure 4D) and accumulation of cells with 53BP1 foci (Figure 4F) and micronuclei (Figure 4G) was reproduced from Jones et al, EMBO.DOI:http://dx.doi.org/10.7554/eLife.18020.011
Mentions: Besides p21, novel negative regulators of alt DNA pols have been recently identified (Bertolin et al., 2015). One of them, USP1, has the ability to remove the ubiquitin moiety from PCNA (Huang et al., 2006; Niimi et al., 2008). Mono-ubiquitinated PCNA interacts with the UBM and UBZ domains of alternative DNA pols favouring their loading to the replisomes (Bienko et al., 2005; Kannouche and Lehmann, 2004; Plosky et al., 2006). During unperturbed replication, PCNA ubiquitination is limited by USP1 (Huang et al., 2006; Niimi et al., 2008), as evidenced by increased PCNA ubiquitination upon USP1 depletion (Figure 4A and B). In constrast, the level of PCNA ubiquitination was not modified when p21 was depleted (Figure 4A). Hence, p21 and USP1 regulate the recruitment of alt pols by distinct mechanisms (Figure 4C). Intriguingly, despite such mechanistic differences, both p21 and USP1 depletion caused a similar effect (both in quality and extent) on nascent DNA elongation (Figure 4D), origin frequency (Figure 4E), the accumulation of cells with 53BP1 foci (Figure 4F) and the number of binucleated cells with MN (Figure 4G). These results reinforce the function of p21 as a negative regulator of alt DNA pols. Moreover, mechanistically distinct regulators of alt DNA pols are equally required to preserve DNA replication and genomic stability during unperturbed replication.10.7554/eLife.18020.011Figure 4.Mechanistically distinct regulators of alt DNA pols are required to facilitate unperturbed DNA replication.

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.