Limits...
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

p21 depletion promotes replication stress and genomic instability in primary human cells.(A and B) IdU track length in Human Foreskin fibroblasts (HFF) and Umbilical Cord Mesenchymal Stem cells (MSC). 100 fiber/sample were analysed in three independent experiments. Western blot showing p21 depletion in the indicated cell line. (C) Representative fibers in siLuc and sip21 HFF cells. (D) Origin frequency in HFF cells. 200 fiber/sample were analyzed in three independent experiments. (E) Cells with more than 5 53BP1 foci were analyzed in MSC cells. 200 nuclei/sample were analyzed in three independent experiments. (F) Representative images of 53BP1 in siLuc and sip21 MSC cells. (G and H) Quantification of MN accumulation. 200 binucleated cells were analyzed in HFF and MSC cells respectively in three independent experiments. (I) Representative images of Binulceated cells with and without MN.DOI:http://dx.doi.org/10.7554/eLife.18020.019
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120883&req=5

fig9: p21 depletion promotes replication stress and genomic instability in primary human cells.(A and B) IdU track length in Human Foreskin fibroblasts (HFF) and Umbilical Cord Mesenchymal Stem cells (MSC). 100 fiber/sample were analysed in three independent experiments. Western blot showing p21 depletion in the indicated cell line. (C) Representative fibers in siLuc and sip21 HFF cells. (D) Origin frequency in HFF cells. 200 fiber/sample were analyzed in three independent experiments. (E) Cells with more than 5 53BP1 foci were analyzed in MSC cells. 200 nuclei/sample were analyzed in three independent experiments. (F) Representative images of 53BP1 in siLuc and sip21 MSC cells. (G and H) Quantification of MN accumulation. 200 binucleated cells were analyzed in HFF and MSC cells respectively in three independent experiments. (I) Representative images of Binulceated cells with and without MN.DOI:http://dx.doi.org/10.7554/eLife.18020.019

Mentions: Given that our results indicate a novel antioncogenic role of p21 in the promotion of DNA replication it was important to determine whether this phenotype was not limited to cancer cells. To address such question we used primary cells from two independent sources: (a) human foreskin fibroblast (HFF) and (b) mesenchyimal stem cells isolated from umbilical cord (MSC). As shown in Figure 9A and B, transfection of p21 siRNA efficiently depleted p21 from both cell types. p21 elimination caused a reduction in the elongation of nascent DNA (Figure 9A–C), which was accompanied with an increase in origin firing (Figure 9D). In turn, such alteration in DNA replication parameters correlated with the accumulation of cells with 53BP1 foci (Figure 9E–F) and micronuclei (Figure 9 G–I). Hence, we postulate that p21 regulates protein-complex formation at the replisomes, promoting the choice of the most adequate polymerase and therefore protecting DNA replication homeostasis. Such novel function of p21 depends exclusively on its ability to interact with PCNA and is needed at every S phase to ensure the accurate finalization of DNA replication (see model in Figure 10).10.7554/eLife.18020.019Figure 9.p21 depletion promotes replication stress and genomic instability in primary human cells.


Cyclin Kinase-independent role of p21 CDKN1A in the promotion of nascent DNA elongation in unstressed cells
p21 depletion promotes replication stress and genomic instability in primary human cells.(A and B) IdU track length in Human Foreskin fibroblasts (HFF) and Umbilical Cord Mesenchymal Stem cells (MSC). 100 fiber/sample were analysed in three independent experiments. Western blot showing p21 depletion in the indicated cell line. (C) Representative fibers in siLuc and sip21 HFF cells. (D) Origin frequency in HFF cells. 200 fiber/sample were analyzed in three independent experiments. (E) Cells with more than 5 53BP1 foci were analyzed in MSC cells. 200 nuclei/sample were analyzed in three independent experiments. (F) Representative images of 53BP1 in siLuc and sip21 MSC cells. (G and H) Quantification of MN accumulation. 200 binucleated cells were analyzed in HFF and MSC cells respectively in three independent experiments. (I) Representative images of Binulceated cells with and without MN.DOI:http://dx.doi.org/10.7554/eLife.18020.019
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120883&req=5

fig9: p21 depletion promotes replication stress and genomic instability in primary human cells.(A and B) IdU track length in Human Foreskin fibroblasts (HFF) and Umbilical Cord Mesenchymal Stem cells (MSC). 100 fiber/sample were analysed in three independent experiments. Western blot showing p21 depletion in the indicated cell line. (C) Representative fibers in siLuc and sip21 HFF cells. (D) Origin frequency in HFF cells. 200 fiber/sample were analyzed in three independent experiments. (E) Cells with more than 5 53BP1 foci were analyzed in MSC cells. 200 nuclei/sample were analyzed in three independent experiments. (F) Representative images of 53BP1 in siLuc and sip21 MSC cells. (G and H) Quantification of MN accumulation. 200 binucleated cells were analyzed in HFF and MSC cells respectively in three independent experiments. (I) Representative images of Binulceated cells with and without MN.DOI:http://dx.doi.org/10.7554/eLife.18020.019
Mentions: Given that our results indicate a novel antioncogenic role of p21 in the promotion of DNA replication it was important to determine whether this phenotype was not limited to cancer cells. To address such question we used primary cells from two independent sources: (a) human foreskin fibroblast (HFF) and (b) mesenchyimal stem cells isolated from umbilical cord (MSC). As shown in Figure 9A and B, transfection of p21 siRNA efficiently depleted p21 from both cell types. p21 elimination caused a reduction in the elongation of nascent DNA (Figure 9A–C), which was accompanied with an increase in origin firing (Figure 9D). In turn, such alteration in DNA replication parameters correlated with the accumulation of cells with 53BP1 foci (Figure 9E–F) and micronuclei (Figure 9 G–I). Hence, we postulate that p21 regulates protein-complex formation at the replisomes, promoting the choice of the most adequate polymerase and therefore protecting DNA replication homeostasis. Such novel function of p21 depends exclusively on its ability to interact with PCNA and is needed at every S phase to ensure the accurate finalization of DNA replication (see model in Figure 10).10.7554/eLife.18020.019Figure 9.p21 depletion promotes replication stress and genomic instability in primary human 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