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A novel role for the mono-ADP-ribosyltransferase PARP14/ARTD8 in promoting homologous recombination and protecting against replication stress.

Nicolae CM, Aho ER, Choe KN, Constantin D, Hu HJ, Lee D, Myung K, Moldovan GL - Nucleic Acids Res. (2015)

Bottom Line: We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites.PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks.Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.

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PARP14 knockdown sensitizes cells to replication fork stalling agents. (A) Quantitative RT-PCR experiments, showing that PARP14 siRNA oligonucleotides efficiently downregulate PARP14 mRNA in HeLa cells. Bars represent average of three experiments, error bars are standard deviations. (B) Western blot with anti-PARP14 antibody showing that PARP14 protein levels are also efficiently downregulated by siRNA in HeLa cells. (C, D) Representative micrographs and quantification of the percentage of HeLa cells with strong γH2AX staining following exposure to HU (1 mM for 12 h) or UV (20 J/m2, analyzed 2 h later). The average of three independent experiments is shown, error bars representing standard errors. No significant difference between control and PARP14-depleted cells was observed in the absence of DNA damage (Supplementary Figure S4A). (E) BrdU/PI double staining and bi-dimensional flow cytometry were performed in control, or PARP14-knockdown HeLa cells. Cycling as well as HU-arrest (2 mM for 18 h)/release samples were analyzed as indicated. An example of flow cytometry plot is shown, indicating the regions that were quantified: R1 = G1 cells (BrdU-negative, 2N DNA content); R2 = early S-phase cells (BrdU-positive, 2N DNA content); R3 = mid-S-phase cells (BrdU-positive, DNA content between 2N and 4N); R4 = late S-phase cells (BrdU-positive, 4N DNA content); R5 = G2 cells (BrdU-negative, 4N DNA content); R6 = S-phase-arrested cells (BrdU-negative, DNA content between 2N and 4N). The graphs show the quantification of R1-R6 regions, averaged from three independent experiments, with standard deviations indicated as error bars. Representative flow plots are shown in Supplementary Figure S5. (F) The percentage of 2N DNA containing cells (R1+R2), 4N DNA containing cells (R4+R5) and of BrdU positive cells (R2+R3+R4) is shown (averaged from three independent experiments, ± standard deviations). P-values are also shown (ns = not significant). (G) Clonogenic survival assay showing that PARP14-knockdown HeLa cells are sensitive to HU treatment. Cells were treated with indicated doses of HU for 48 h, then incubated in fresh, drug-free media for 2 weeks. The average of three independent experiments, with standard deviations as error bars, is shown. Similar results were obtained in 8988T cells (Supplementary Figure S7). (H) Representative clonogenic assay showing HU sensitivity of PARP14-depleted HeLa cells. (I) UV (200 J/m2) -induced loss of viability is increased in the absence of PARP14. Left: representative micrographs showing accumulation of cells with aberrant multi-lobed nuclei (pre-apoptotic cells) 2 h after UV irradiation. HeLa cells were analyzed by DAPI staining. Right: quantification of at least three experiments. Shown are averages, and error bars represent standard errors. At least 25 cells were counted.
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Figure 2: PARP14 knockdown sensitizes cells to replication fork stalling agents. (A) Quantitative RT-PCR experiments, showing that PARP14 siRNA oligonucleotides efficiently downregulate PARP14 mRNA in HeLa cells. Bars represent average of three experiments, error bars are standard deviations. (B) Western blot with anti-PARP14 antibody showing that PARP14 protein levels are also efficiently downregulated by siRNA in HeLa cells. (C, D) Representative micrographs and quantification of the percentage of HeLa cells with strong γH2AX staining following exposure to HU (1 mM for 12 h) or UV (20 J/m2, analyzed 2 h later). The average of three independent experiments is shown, error bars representing standard errors. No significant difference between control and PARP14-depleted cells was observed in the absence of DNA damage (Supplementary Figure S4A). (E) BrdU/PI double staining and bi-dimensional flow cytometry were performed in control, or PARP14-knockdown HeLa cells. Cycling as well as HU-arrest (2 mM for 18 h)/release samples were analyzed as indicated. An example of flow cytometry plot is shown, indicating the regions that were quantified: R1 = G1 cells (BrdU-negative, 2N DNA content); R2 = early S-phase cells (BrdU-positive, 2N DNA content); R3 = mid-S-phase cells (BrdU-positive, DNA content between 2N and 4N); R4 = late S-phase cells (BrdU-positive, 4N DNA content); R5 = G2 cells (BrdU-negative, 4N DNA content); R6 = S-phase-arrested cells (BrdU-negative, DNA content between 2N and 4N). The graphs show the quantification of R1-R6 regions, averaged from three independent experiments, with standard deviations indicated as error bars. Representative flow plots are shown in Supplementary Figure S5. (F) The percentage of 2N DNA containing cells (R1+R2), 4N DNA containing cells (R4+R5) and of BrdU positive cells (R2+R3+R4) is shown (averaged from three independent experiments, ± standard deviations). P-values are also shown (ns = not significant). (G) Clonogenic survival assay showing that PARP14-knockdown HeLa cells are sensitive to HU treatment. Cells were treated with indicated doses of HU for 48 h, then incubated in fresh, drug-free media for 2 weeks. The average of three independent experiments, with standard deviations as error bars, is shown. Similar results were obtained in 8988T cells (Supplementary Figure S7). (H) Representative clonogenic assay showing HU sensitivity of PARP14-depleted HeLa cells. (I) UV (200 J/m2) -induced loss of viability is increased in the absence of PARP14. Left: representative micrographs showing accumulation of cells with aberrant multi-lobed nuclei (pre-apoptotic cells) 2 h after UV irradiation. HeLa cells were analyzed by DAPI staining. Right: quantification of at least three experiments. Shown are averages, and error bars represent standard errors. At least 25 cells were counted.

Mentions: We next investigated how PARP14 depletion impacts the response to DNA damage. Two different siRNA oligonucleotides were able to efficiently reduce the levels of PARP14 in HeLa, U2OS and 8988T cells (Figure 2A and B and Supplementary Figure S3). PARP14 knockdown in HeLa cells resulted in increased γH2AX staining following exposure to replication fork stalling agents HU and UV (Figure 2C and D), while no difference was observed in the absence of drug treatment (Supplementary Figure S4A). Similar results were obtained in U2OS cells (Supplementary Figure S4B and C). These results suggest an inability to process stalled replication forks in the absence of PARP14. We next monitored cell cycle distribution in control and PARP14-knockdown HeLa cells. We treated HeLa cells with BrdU and performed anti-BrdU and PI double staining followed by bi-dimensional flow cytometry (Figure 2E and F and Supplementary Figure S5). The percentage of BrdU-positive cells and the general cell cycle distribution were not affected by PARP14 knockdown in the absence of drug treatment. We next treated cells with 2-mM HU for 18h and analyzed them at several time points following drug removal. PARP14 knockdown cells showed a reduced percentage of cells in mid-S-phase 3 hours after release, and a reduced percentage of cells in late S-phase 6 hours after release (Figure 2E and Supplementary Figure S5). The percentage of cells with replicated DNA (4N DNA content) was significantly reduced in PARP14 knockdown cells 3 and 6 h after drug removal (Figure 2F and Supplementary Figure S6). These results further indicate that PARP14 knockdown cells have a significant defect in progression through S-phase under replication stress conditions. Indeed, we found that PARP14-depleted cells were sensitive to HU in clonogenic survival assays (Figure 2G and H and Supplementary Figure S7). Moreover, we observed an increase in aberrant, multi-lobed pre-apoptotic nuclei in PARP14 knockdown HeLa cells after UV exposure, suggesting that PARP14 is important for protecting cells against UV damage (Figure 2I). Altogether, these results show that PARP14 is important for DNA replication under stress conditions.


A novel role for the mono-ADP-ribosyltransferase PARP14/ARTD8 in promoting homologous recombination and protecting against replication stress.

Nicolae CM, Aho ER, Choe KN, Constantin D, Hu HJ, Lee D, Myung K, Moldovan GL - Nucleic Acids Res. (2015)

PARP14 knockdown sensitizes cells to replication fork stalling agents. (A) Quantitative RT-PCR experiments, showing that PARP14 siRNA oligonucleotides efficiently downregulate PARP14 mRNA in HeLa cells. Bars represent average of three experiments, error bars are standard deviations. (B) Western blot with anti-PARP14 antibody showing that PARP14 protein levels are also efficiently downregulated by siRNA in HeLa cells. (C, D) Representative micrographs and quantification of the percentage of HeLa cells with strong γH2AX staining following exposure to HU (1 mM for 12 h) or UV (20 J/m2, analyzed 2 h later). The average of three independent experiments is shown, error bars representing standard errors. No significant difference between control and PARP14-depleted cells was observed in the absence of DNA damage (Supplementary Figure S4A). (E) BrdU/PI double staining and bi-dimensional flow cytometry were performed in control, or PARP14-knockdown HeLa cells. Cycling as well as HU-arrest (2 mM for 18 h)/release samples were analyzed as indicated. An example of flow cytometry plot is shown, indicating the regions that were quantified: R1 = G1 cells (BrdU-negative, 2N DNA content); R2 = early S-phase cells (BrdU-positive, 2N DNA content); R3 = mid-S-phase cells (BrdU-positive, DNA content between 2N and 4N); R4 = late S-phase cells (BrdU-positive, 4N DNA content); R5 = G2 cells (BrdU-negative, 4N DNA content); R6 = S-phase-arrested cells (BrdU-negative, DNA content between 2N and 4N). The graphs show the quantification of R1-R6 regions, averaged from three independent experiments, with standard deviations indicated as error bars. Representative flow plots are shown in Supplementary Figure S5. (F) The percentage of 2N DNA containing cells (R1+R2), 4N DNA containing cells (R4+R5) and of BrdU positive cells (R2+R3+R4) is shown (averaged from three independent experiments, ± standard deviations). P-values are also shown (ns = not significant). (G) Clonogenic survival assay showing that PARP14-knockdown HeLa cells are sensitive to HU treatment. Cells were treated with indicated doses of HU for 48 h, then incubated in fresh, drug-free media for 2 weeks. The average of three independent experiments, with standard deviations as error bars, is shown. Similar results were obtained in 8988T cells (Supplementary Figure S7). (H) Representative clonogenic assay showing HU sensitivity of PARP14-depleted HeLa cells. (I) UV (200 J/m2) -induced loss of viability is increased in the absence of PARP14. Left: representative micrographs showing accumulation of cells with aberrant multi-lobed nuclei (pre-apoptotic cells) 2 h after UV irradiation. HeLa cells were analyzed by DAPI staining. Right: quantification of at least three experiments. Shown are averages, and error bars represent standard errors. At least 25 cells were counted.
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Figure 2: PARP14 knockdown sensitizes cells to replication fork stalling agents. (A) Quantitative RT-PCR experiments, showing that PARP14 siRNA oligonucleotides efficiently downregulate PARP14 mRNA in HeLa cells. Bars represent average of three experiments, error bars are standard deviations. (B) Western blot with anti-PARP14 antibody showing that PARP14 protein levels are also efficiently downregulated by siRNA in HeLa cells. (C, D) Representative micrographs and quantification of the percentage of HeLa cells with strong γH2AX staining following exposure to HU (1 mM for 12 h) or UV (20 J/m2, analyzed 2 h later). The average of three independent experiments is shown, error bars representing standard errors. No significant difference between control and PARP14-depleted cells was observed in the absence of DNA damage (Supplementary Figure S4A). (E) BrdU/PI double staining and bi-dimensional flow cytometry were performed in control, or PARP14-knockdown HeLa cells. Cycling as well as HU-arrest (2 mM for 18 h)/release samples were analyzed as indicated. An example of flow cytometry plot is shown, indicating the regions that were quantified: R1 = G1 cells (BrdU-negative, 2N DNA content); R2 = early S-phase cells (BrdU-positive, 2N DNA content); R3 = mid-S-phase cells (BrdU-positive, DNA content between 2N and 4N); R4 = late S-phase cells (BrdU-positive, 4N DNA content); R5 = G2 cells (BrdU-negative, 4N DNA content); R6 = S-phase-arrested cells (BrdU-negative, DNA content between 2N and 4N). The graphs show the quantification of R1-R6 regions, averaged from three independent experiments, with standard deviations indicated as error bars. Representative flow plots are shown in Supplementary Figure S5. (F) The percentage of 2N DNA containing cells (R1+R2), 4N DNA containing cells (R4+R5) and of BrdU positive cells (R2+R3+R4) is shown (averaged from three independent experiments, ± standard deviations). P-values are also shown (ns = not significant). (G) Clonogenic survival assay showing that PARP14-knockdown HeLa cells are sensitive to HU treatment. Cells were treated with indicated doses of HU for 48 h, then incubated in fresh, drug-free media for 2 weeks. The average of three independent experiments, with standard deviations as error bars, is shown. Similar results were obtained in 8988T cells (Supplementary Figure S7). (H) Representative clonogenic assay showing HU sensitivity of PARP14-depleted HeLa cells. (I) UV (200 J/m2) -induced loss of viability is increased in the absence of PARP14. Left: representative micrographs showing accumulation of cells with aberrant multi-lobed nuclei (pre-apoptotic cells) 2 h after UV irradiation. HeLa cells were analyzed by DAPI staining. Right: quantification of at least three experiments. Shown are averages, and error bars represent standard errors. At least 25 cells were counted.
Mentions: We next investigated how PARP14 depletion impacts the response to DNA damage. Two different siRNA oligonucleotides were able to efficiently reduce the levels of PARP14 in HeLa, U2OS and 8988T cells (Figure 2A and B and Supplementary Figure S3). PARP14 knockdown in HeLa cells resulted in increased γH2AX staining following exposure to replication fork stalling agents HU and UV (Figure 2C and D), while no difference was observed in the absence of drug treatment (Supplementary Figure S4A). Similar results were obtained in U2OS cells (Supplementary Figure S4B and C). These results suggest an inability to process stalled replication forks in the absence of PARP14. We next monitored cell cycle distribution in control and PARP14-knockdown HeLa cells. We treated HeLa cells with BrdU and performed anti-BrdU and PI double staining followed by bi-dimensional flow cytometry (Figure 2E and F and Supplementary Figure S5). The percentage of BrdU-positive cells and the general cell cycle distribution were not affected by PARP14 knockdown in the absence of drug treatment. We next treated cells with 2-mM HU for 18h and analyzed them at several time points following drug removal. PARP14 knockdown cells showed a reduced percentage of cells in mid-S-phase 3 hours after release, and a reduced percentage of cells in late S-phase 6 hours after release (Figure 2E and Supplementary Figure S5). The percentage of cells with replicated DNA (4N DNA content) was significantly reduced in PARP14 knockdown cells 3 and 6 h after drug removal (Figure 2F and Supplementary Figure S6). These results further indicate that PARP14 knockdown cells have a significant defect in progression through S-phase under replication stress conditions. Indeed, we found that PARP14-depleted cells were sensitive to HU in clonogenic survival assays (Figure 2G and H and Supplementary Figure S7). Moreover, we observed an increase in aberrant, multi-lobed pre-apoptotic nuclei in PARP14 knockdown HeLa cells after UV exposure, suggesting that PARP14 is important for protecting cells against UV damage (Figure 2I). Altogether, these results show that PARP14 is important for DNA replication under stress conditions.

Bottom Line: We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites.PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks.Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.

Show MeSH
Related in: MedlinePlus