RESUMEN
Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.
Asunto(s)
Apoptosis/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/deficiencia , ARN Polimerasas Dirigidas por ADN/metabolismo , Células Eucariotas/enzimología , Dímeros de Pirimidina/metabolismo , ARN/biosíntesis , Animales , Apoptosis/efectos de la radiación , Células CHO , Cricetinae , ADN Helicasas , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , ARN Polimerasas Dirigidas por ADN/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/genética , Relación Dosis-Respuesta en la Radiación , Células Eucariotas/efectos de la radiación , Mutación/genética , Dímeros de Pirimidina/antagonistas & inhibidores , ARN/genética , Rayos UltravioletaRESUMEN
Photolyase absorbs blue light and employs the energy to remove UV-induced DNA damage, cyclobutane pyrimidine dimers, or pyrimidine pyrimidone (6-4) lesions. These enzymes have been found in many living organisms ranging from bacteria to aplacental mammals, but their photoreactivation effect, such as survival increase of UV-irradiated cells by light-illumination, has not been identified in placental mammals, including humans. Therefore, we introduced a photolyase gene derived from the marsupial rat kangaroo, Potorous tridactylus, into HeLa cells and established the first human cell line capable of photorepairing UV-induced pyrimidine dimers. Several clones were found to increase cell survival after UV irradiation when illuminated by fluorescent light. The induction of apoptosis by UV irradiation was investigated in these photoreactivation-proficient cells. Several typical features of the programmed cell death, such as internucleosomal DNA degradation, presence of subdiploid cells, loss of membrane integrity, and chromosomal condensation, were found to be induced by UV in the HeLa cells, but they can be reduced by photorepair. This implicates that cyclobutane pyrimidine dimers cause UV-induced apoptosis in human cells.