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.
Highlights
Ribosomal RNA is synthesized in the nucleolus by RNA polymerase 1 (Pol1)
Several proteins involved in DNA damage repair including poly(ADP-ribose) polymerase 1 (PARP-1), the DNA-dependent protein kinase (DNA-PK) subunit Ku, WRN and
ethynyl uridine (EU) accumulation in the nucleoli was blocked by the Pol1 inhibitor actinomycin D (Supplementary Figure S2)
Summary
Ribosomal RNA (rRNA) is synthesized in the nucleolus by RNA polymerase 1 (Pol). Pol activity is dependent on the assembly of many separate proteins on rRNA gene promoters. Several proteins involved in DNA damage repair including poly(ADP-ribose) polymerase 1 (PARP-1), the DNA-dependent protein kinase (DNA-PK) subunit Ku (a heterodimer composed of Ku70 and Ku86), WRN and SSRP1 are present in the nucleolus and relocate to the nucleoplasm after damage [5,6,7,8,9]. PARP-1 forms a complex with DNA-PK and the facilitator of chromatin transcription, FACT [24,25]. These reports suggest a role for both DNA-PK and PARP-1 in rRNA synthesis following DNA damage
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