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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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Cisplatin-induced PARP-1 activity is DNA-PK dependent. (A) Immunofluorescence after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Representative fields stained for DAPI and DNA-PKcs p-Ser2056 (p-Ser). (B) Quantification of the average p-Ser fluorescence per nucleus from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. (D) Immunofluorescence of PAR after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Supplementary Figure S13 describes image production; the original image (orig) and the processed image (proj) are shown. (E) Quantification of the average relative number of PAR foci per nucleus is shown from experiments using wortmannin and olaparib, (F) Nu7026 and Nu7441 or (G) in cells expressing either nonsilencing or DNA-PKcs shRNA. Each condition was normalized to cells treated with DMSO but not cisplatin. One-way ANOVA was followed by Holm–Sidak’s test to compare responses to cisplatin of cells exposed to wortmannin or olaparib or expressing DNA-PKcs shRNA versus cells exposed to DMSO or expressing NS shRNA. *, ** represent P ≤ 0.05, 0.01, respectively; ns indicates nonsignificant result (P > 0.05).
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gkt502-F3: Cisplatin-induced PARP-1 activity is DNA-PK dependent. (A) Immunofluorescence after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Representative fields stained for DAPI and DNA-PKcs p-Ser2056 (p-Ser). (B) Quantification of the average p-Ser fluorescence per nucleus from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. (D) Immunofluorescence of PAR after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Supplementary Figure S13 describes image production; the original image (orig) and the processed image (proj) are shown. (E) Quantification of the average relative number of PAR foci per nucleus is shown from experiments using wortmannin and olaparib, (F) Nu7026 and Nu7441 or (G) in cells expressing either nonsilencing or DNA-PKcs shRNA. Each condition was normalized to cells treated with DMSO but not cisplatin. One-way ANOVA was followed by Holm–Sidak’s test to compare responses to cisplatin of cells exposed to wortmannin or olaparib or expressing DNA-PKcs shRNA versus cells exposed to DMSO or expressing NS shRNA. *, ** represent P ≤ 0.05, 0.01, respectively; ns indicates nonsignificant result (P > 0.05).

Mentions: We measured levels of auto-phosphorylation of DNA-PKcs at serine 2056 (DNA-PKcs p-Ser2056) by immunofluorescence before and after cisplatin treatment. Twenty-two hours after cisplatin treatment, we observed a significant signal increase of DNA-PKcs p-Ser2056 inside nuclei (Figure 3A). DNA-PK activation was significantly inhibited in cells pretreated with wortmannin and Nu7026 (Figure 3B–C). Confirmation of inhibition of DNA-PK by Nu7441 required western blotting analysis (Supplementary Figure S21). Olaparib pretreatment had no effect on DNA-PK activation (Figure 3A and B), which was verified by western blotting (Figure 4A). These results suggest PARP-1 does not affect DNA-PK activation after DNA damage. Interestingly, PARP-1 inhibition leads to activation of DNA-PK in BRCA2-deficient cells but not in cells with wild-type BRCA2 (41).Figure 3.


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

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

Cisplatin-induced PARP-1 activity is DNA-PK dependent. (A) Immunofluorescence after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Representative fields stained for DAPI and DNA-PKcs p-Ser2056 (p-Ser). (B) Quantification of the average p-Ser fluorescence per nucleus from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. (D) Immunofluorescence of PAR after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Supplementary Figure S13 describes image production; the original image (orig) and the processed image (proj) are shown. (E) Quantification of the average relative number of PAR foci per nucleus is shown from experiments using wortmannin and olaparib, (F) Nu7026 and Nu7441 or (G) in cells expressing either nonsilencing or DNA-PKcs shRNA. Each condition was normalized to cells treated with DMSO but not cisplatin. One-way ANOVA was followed by Holm–Sidak’s test to compare responses to cisplatin of cells exposed to wortmannin or olaparib or expressing DNA-PKcs shRNA versus cells exposed to DMSO or expressing NS shRNA. *, ** represent P ≤ 0.05, 0.01, respectively; ns indicates nonsignificant result (P > 0.05).
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gkt502-F3: Cisplatin-induced PARP-1 activity is DNA-PK dependent. (A) Immunofluorescence after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Representative fields stained for DAPI and DNA-PKcs p-Ser2056 (p-Ser). (B) Quantification of the average p-Ser fluorescence per nucleus from experiments using wortmannin and olaparib or (C) Nu7026 and Nu7441. (D) Immunofluorescence of PAR after treatment with cisplatin and wortmannin or olaparib as described in Figure 2. Supplementary Figure S13 describes image production; the original image (orig) and the processed image (proj) are shown. (E) Quantification of the average relative number of PAR foci per nucleus is shown from experiments using wortmannin and olaparib, (F) Nu7026 and Nu7441 or (G) in cells expressing either nonsilencing or DNA-PKcs shRNA. Each condition was normalized to cells treated with DMSO but not cisplatin. One-way ANOVA was followed by Holm–Sidak’s test to compare responses to cisplatin of cells exposed to wortmannin or olaparib or expressing DNA-PKcs shRNA versus cells exposed to DMSO or expressing NS shRNA. *, ** represent P ≤ 0.05, 0.01, respectively; ns indicates nonsignificant result (P > 0.05).
Mentions: We measured levels of auto-phosphorylation of DNA-PKcs at serine 2056 (DNA-PKcs p-Ser2056) by immunofluorescence before and after cisplatin treatment. Twenty-two hours after cisplatin treatment, we observed a significant signal increase of DNA-PKcs p-Ser2056 inside nuclei (Figure 3A). DNA-PK activation was significantly inhibited in cells pretreated with wortmannin and Nu7026 (Figure 3B–C). Confirmation of inhibition of DNA-PK by Nu7441 required western blotting analysis (Supplementary Figure S21). Olaparib pretreatment had no effect on DNA-PK activation (Figure 3A and B), which was verified by western blotting (Figure 4A). These results suggest PARP-1 does not affect DNA-PK activation after DNA damage. Interestingly, PARP-1 inhibition leads to activation of DNA-PK in BRCA2-deficient cells but not in cells with wild-type BRCA2 (41).Figure 3.

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