RESUMO
Environmental agents are constantly challenging cells by damaging DNA, leading to the blockage of transcription elongation. How do cells deal with transcription-blockage and how is transcription restarted after the blocking lesions are removed? Here we review the processes responsible for the removal of transcription-blocking lesions, as well as mechanisms of transcription restart. We also discuss recent data suggesting that blocked RNA polymerases may not resume transcription from the site of the lesion following its removal but, rather, are forced to start over from the beginning of genes.
Assuntos
Transcrição Gênica , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , RNA/biossíntese , RNA/genética , Elongação da Transcrição Genética/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5'-3' direction with slow recovery and TC-NER at the 3' end of long genes. RNA synthesis resumed fully at the 3'-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases.
Assuntos
Reparo do DNA , Fibroblastos/efeitos da radiação , RNA/genética , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Células Cultivadas , Criança , Pré-Escolar , DNA/genética , DNA/metabolismo , Dano ao DNA , Replicação do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Masculino , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , Fatores de Tempo , Transcriptoma/efeitos da radiaçãoRESUMO
Ultraviolet (UV) light can stall replication forks owing to the formation of bulky lesions in the DNA. Replication across these blocking lesions occurs through translesion DNA synthesis, and cells activate the ATR damage responses to UV. However, it remains unclear whether lesion bypass requires the replication checkpoint because ATR is not necessary for PCNA ubiquitylation. We observed that ATR knockdown by siRNA increased replication stress and promoted early induction of apoptosis following UVB irradiation in SV40-immortalized human cells, including cells from XP-V and XP-C patients. XP-V cells were further sensitized by the silencing, indicating that DNA polymerase η (Pol η) remains active despite ATR control. However, following UVB irradiation, ATR-depleted cells were unable to achieve mitosis, as would be expected after the loss of a DNA checkpoint control. Thus, ATR also regulates replication arrest recovery following UVB-induced damage, independently of Pol η, in SV40-immortalized cell lines. The ATR-mediated DNA damage response regulates replication and different tolerance pathways, and in these cells, ATR depletion induces replication catastrophe, which contributes to explain the potential of ATR inhibition to protect against UVB-induced carcinogenesis.
