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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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Regulation of rRNA synthesis is distinct from DNA synthesis. (A) Immunofluorescence of BrdU incorporation was performed in cells treated with cisplatin, wortmannin and olaparib as in Figure 2. Representative fields stained for DAPI and BrdU. (B) Quantification of BrdU incorporation expressed as ratios of total cells actively replicating DNA from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. Each condition was normalized to cells treated with DMSO or indicated inhibitors without cisplatin treatment. Decrease in nuclear BrdU significantly differs from the baseline after two-way ANOVA test followed by Dunnett’s test (P ≤ 0.001), while cisplatin-treated values were determined by separate two-way ANOVA followed by Holm–Sidak’s test (P > 0.05, ns). (D) Histograms plotting the area of propidium iodide staining (PI) and number of cells as determined by flow cytometry. G1, G2 and S phases were shaded by ModFit. Percentage of cells in G1, G2 and S phases is included as a table. (E) Schematic depicting the involvement of DNA-PK and PARP-1 in DNA damage-induced inhibition of rRNA synthesis.
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gkt502-F5: Regulation of rRNA synthesis is distinct from DNA synthesis. (A) Immunofluorescence of BrdU incorporation was performed in cells treated with cisplatin, wortmannin and olaparib as in Figure 2. Representative fields stained for DAPI and BrdU. (B) Quantification of BrdU incorporation expressed as ratios of total cells actively replicating DNA from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. Each condition was normalized to cells treated with DMSO or indicated inhibitors without cisplatin treatment. Decrease in nuclear BrdU significantly differs from the baseline after two-way ANOVA test followed by Dunnett’s test (P ≤ 0.001), while cisplatin-treated values were determined by separate two-way ANOVA followed by Holm–Sidak’s test (P > 0.05, ns). (D) Histograms plotting the area of propidium iodide staining (PI) and number of cells as determined by flow cytometry. G1, G2 and S phases were shaded by ModFit. Percentage of cells in G1, G2 and S phases is included as a table. (E) Schematic depicting the involvement of DNA-PK and PARP-1 in DNA damage-induced inhibition of rRNA synthesis.

Mentions: Inhibition of rRNA synthesis 24 h after DNA damage could be due to a cell cycle effect. Both the cell cycle and DNA replication are inhibited by genotoxic stress (42). We monitored BrdU incorporation in situ by adding BrdU 1 h before fixation to determine the fraction of cells with ongoing DNA replication. Twenty-two hours after the cisplatin pulse, DNA replication was significantly reduced. To determine if inhibition of PARP-1 or DNA-PK induced resumption of the cell cycle, cells were treated with cisplatin and inhibitors as in Figure 2. In contrast to rRNA synthesis, DNA replication was blocked even with inhibition of PARP-1 and DNA-PK (Figure 5A–C). To determine in which phase of the cell cycle cisplatin treatment induced arrest, we performed flow cytometry. As previously described by Sorenson and Eastman, we found cells treated with cisplatin arrested in S phase (43). Pretreatment with inhibitors did not change the phase at which arrest occurred (Figure 5D and Supplementary Figure S24). Cisplatin-induced block of rRNA synthesis is prevented by DNA-PK and PARP-1 inhibition without altering cell cycle withdrawal in S phase. Thus, the control of rRNA synthesis by DNA-PK and PARP-1 after DNA damage is uncoupled from regulation of the cell cycle (Figure 5E).Figure 5.


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

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

Regulation of rRNA synthesis is distinct from DNA synthesis. (A) Immunofluorescence of BrdU incorporation was performed in cells treated with cisplatin, wortmannin and olaparib as in Figure 2. Representative fields stained for DAPI and BrdU. (B) Quantification of BrdU incorporation expressed as ratios of total cells actively replicating DNA from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. Each condition was normalized to cells treated with DMSO or indicated inhibitors without cisplatin treatment. Decrease in nuclear BrdU significantly differs from the baseline after two-way ANOVA test followed by Dunnett’s test (P ≤ 0.001), while cisplatin-treated values were determined by separate two-way ANOVA followed by Holm–Sidak’s test (P > 0.05, ns). (D) Histograms plotting the area of propidium iodide staining (PI) and number of cells as determined by flow cytometry. G1, G2 and S phases were shaded by ModFit. Percentage of cells in G1, G2 and S phases is included as a table. (E) Schematic depicting the involvement of DNA-PK and PARP-1 in DNA damage-induced inhibition of rRNA synthesis.
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gkt502-F5: Regulation of rRNA synthesis is distinct from DNA synthesis. (A) Immunofluorescence of BrdU incorporation was performed in cells treated with cisplatin, wortmannin and olaparib as in Figure 2. Representative fields stained for DAPI and BrdU. (B) Quantification of BrdU incorporation expressed as ratios of total cells actively replicating DNA from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. Each condition was normalized to cells treated with DMSO or indicated inhibitors without cisplatin treatment. Decrease in nuclear BrdU significantly differs from the baseline after two-way ANOVA test followed by Dunnett’s test (P ≤ 0.001), while cisplatin-treated values were determined by separate two-way ANOVA followed by Holm–Sidak’s test (P > 0.05, ns). (D) Histograms plotting the area of propidium iodide staining (PI) and number of cells as determined by flow cytometry. G1, G2 and S phases were shaded by ModFit. Percentage of cells in G1, G2 and S phases is included as a table. (E) Schematic depicting the involvement of DNA-PK and PARP-1 in DNA damage-induced inhibition of rRNA synthesis.
Mentions: Inhibition of rRNA synthesis 24 h after DNA damage could be due to a cell cycle effect. Both the cell cycle and DNA replication are inhibited by genotoxic stress (42). We monitored BrdU incorporation in situ by adding BrdU 1 h before fixation to determine the fraction of cells with ongoing DNA replication. Twenty-two hours after the cisplatin pulse, DNA replication was significantly reduced. To determine if inhibition of PARP-1 or DNA-PK induced resumption of the cell cycle, cells were treated with cisplatin and inhibitors as in Figure 2. In contrast to rRNA synthesis, DNA replication was blocked even with inhibition of PARP-1 and DNA-PK (Figure 5A–C). To determine in which phase of the cell cycle cisplatin treatment induced arrest, we performed flow cytometry. As previously described by Sorenson and Eastman, we found cells treated with cisplatin arrested in S phase (43). Pretreatment with inhibitors did not change the phase at which arrest occurred (Figure 5D and Supplementary Figure S24). Cisplatin-induced block of rRNA synthesis is prevented by DNA-PK and PARP-1 inhibition without altering cell cycle withdrawal in S phase. Thus, the control of rRNA synthesis by DNA-PK and PARP-1 after DNA damage is uncoupled from regulation of the cell cycle (Figure 5E).Figure 5.

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