RESUMO
The transcription/DNA repair factor TFIIH is organized into a core that associates with the CDK-activating kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during nucleotide excision repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/excision of the damaged oligonucleotide and thereby the repair of the DNA. Following repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Trifosfato de Adenosina/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Humanos , Modelos Biológicos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/efeitos da radiação , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
In this study, different approaches were used to assess and overcome the severe effects of interference from the sample matrix from different types of sludges and wastewater on the analysis of nine beta-blockers and the beta sympathomimetic clenbuterol. The partitioning of the target compounds into sludge was investigated in wastewater treatment plants (WWTPs) in both Canada and Germany to evaluate whether this is an important mechanism for removal from sewage. Due to ion suppression in the electro spray interface, absolute recoveries were for certain compounds even lower than 20%. By using surrogate standards, acceptable relative recoveries of >75% were achieved for WWTP influents and effluents and for sludges. These matrix effects underline the need to use appropriate surrogate standards to aid in analyte quantitation. Using the developed methods, beta-blockers were detected at concentrations up to 2 microg/L in WWTP effluents, with metoprolol, sotalol, and atenolol present as the dominant compounds. Removal rates within WWTPs were highly inconsistent and ranged from 1-69%. Propranolol showed the greatest degree of partitioning into sludge with solid/water partition coefficients of one order of magnitude higher than those for all other compounds. However, even for propranolol, sorption did not contribute significantly to the overall elimination in WWTPs. It is likely that the removal of beta-blockers during waste water treatment can be attributed primarily to microbial biodegradation.