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DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1.

Calkins AS, Iglehart JD, Lazaro JB - Nucleic Acids Res. (2013)

Bottom Line: Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis.However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation.Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

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Inhibition of rRNA synthesis. (A) Nucleolar Ku and PARP-1 are lost after 2 h of treatment with 25 µg/ml cisplatin (Cis). Nuclei were outlined using CellProfiler software. (B) Cells were exposed to 25 µg/ml cisplatin for 2 h and analyzed for EU incorporation at indicated times after cisplatin withdrawal. (C) Inhibition of rRNA synthesis by cisplatin at 10, 25 and 50 µg/ml is shown by EU incorporation. Nucleoli were stained by anti-NOL1. (D) Quantification of adjusted nucleolar EU fluorescence shown in (C). Each condition was normalized to cells without cisplatin treatment. One-way ANOVA was followed by Dunnett’s test for comparison of each dose of cisplatin to the baseline. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively. (E) Schematic of the sequence of steps in assays for RNA synthesis.
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gkt502-F1: Inhibition of rRNA synthesis. (A) Nucleolar Ku and PARP-1 are lost after 2 h of treatment with 25 µg/ml cisplatin (Cis). Nuclei were outlined using CellProfiler software. (B) Cells were exposed to 25 µg/ml cisplatin for 2 h and analyzed for EU incorporation at indicated times after cisplatin withdrawal. (C) Inhibition of rRNA synthesis by cisplatin at 10, 25 and 50 µg/ml is shown by EU incorporation. Nucleoli were stained by anti-NOL1. (D) Quantification of adjusted nucleolar EU fluorescence shown in (C). Each condition was normalized to cells without cisplatin treatment. One-way ANOVA was followed by Dunnett’s test for comparison of each dose of cisplatin to the baseline. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively. (E) Schematic of the sequence of steps in assays for RNA synthesis.

Mentions: As previously reported, 2 h of treatment with cisplatin led to loss of nucleolar Ku (5) and similarly to loss of nucleolar PARP-1 (Figure 1A). For this reason, we analyzed the effect of a 2-h cisplatin pulse on rRNA synthesis. We measured incorporation of EU at various time points following this initial cisplatin treatment. Although no inhibition of RNA synthesis was found during the first 10 h following withdrawal of cisplatin, nucleolar RNA synthesis was blocked 22 h after the cisplatin pulse, concordant with the results of Jordan and Carmo-Fonseca (3) (Figure 1B). This effect was not due to alteration of the overall RNA content in the nucleolus, shown by total RNA staining (Supplementary Figure S3).Figure 1.


DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1.

Calkins AS, Iglehart JD, Lazaro JB - Nucleic Acids Res. (2013)

Inhibition of rRNA synthesis. (A) Nucleolar Ku and PARP-1 are lost after 2 h of treatment with 25 µg/ml cisplatin (Cis). Nuclei were outlined using CellProfiler software. (B) Cells were exposed to 25 µg/ml cisplatin for 2 h and analyzed for EU incorporation at indicated times after cisplatin withdrawal. (C) Inhibition of rRNA synthesis by cisplatin at 10, 25 and 50 µg/ml is shown by EU incorporation. Nucleoli were stained by anti-NOL1. (D) Quantification of adjusted nucleolar EU fluorescence shown in (C). Each condition was normalized to cells without cisplatin treatment. One-way ANOVA was followed by Dunnett’s test for comparison of each dose of cisplatin to the baseline. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively. (E) Schematic of the sequence of steps in assays for RNA synthesis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753630&req=5

gkt502-F1: Inhibition of rRNA synthesis. (A) Nucleolar Ku and PARP-1 are lost after 2 h of treatment with 25 µg/ml cisplatin (Cis). Nuclei were outlined using CellProfiler software. (B) Cells were exposed to 25 µg/ml cisplatin for 2 h and analyzed for EU incorporation at indicated times after cisplatin withdrawal. (C) Inhibition of rRNA synthesis by cisplatin at 10, 25 and 50 µg/ml is shown by EU incorporation. Nucleoli were stained by anti-NOL1. (D) Quantification of adjusted nucleolar EU fluorescence shown in (C). Each condition was normalized to cells without cisplatin treatment. One-way ANOVA was followed by Dunnett’s test for comparison of each dose of cisplatin to the baseline. *, **, *** represent P ≤ 0.05, 0.01, 0.001, respectively. (E) Schematic of the sequence of steps in assays for RNA synthesis.
Mentions: As previously reported, 2 h of treatment with cisplatin led to loss of nucleolar Ku (5) and similarly to loss of nucleolar PARP-1 (Figure 1A). For this reason, we analyzed the effect of a 2-h cisplatin pulse on rRNA synthesis. We measured incorporation of EU at various time points following this initial cisplatin treatment. Although no inhibition of RNA synthesis was found during the first 10 h following withdrawal of cisplatin, nucleolar RNA synthesis was blocked 22 h after the cisplatin pulse, concordant with the results of Jordan and Carmo-Fonseca (3) (Figure 1B). This effect was not due to alteration of the overall RNA content in the nucleolus, shown by total RNA staining (Supplementary Figure S3).Figure 1.

Bottom Line: Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis.However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation.Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA and Department of Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

Show MeSH
Related in: MedlinePlus