RESUMO
DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major actor of both nucleotide excision repair subpathways of which transcription-coupled repair highlights the interplay between DNA repair and transcription. Using an unbiased proteomic approach, we have identified the protein eleven-nineteen lysine-rich leukemia (ELL) as a TFIIH partner. Here we show that ELL is recruited to UV-damaged chromatin in a Cdk7- dependent manner (a component of the cyclin-dependent activating kinase subcomplex of TFIIH). We demonstrate that depletion of ELL strongly hinders RNA polymerase II (RNA Pol II) transcription resumption after lesion removal and DNA gap filling. Lack of ELL was also observed to increase RNA Pol II retention to the chromatin during this process. Identifying ELL as an essential player for RNA Pol II restart during cellular DNA damage response opens the way to obtaining a mechanistic description of transcription resumption after DNA repair.
Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/metabolismo , Ativação Transcricional/fisiologia , Fatores de Elongação da Transcrição/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Clonagem Molecular , Primers do DNA/genética , Recuperação de Fluorescência Após Fotodegradação , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Trichothiodystrophy group A (TTD-A) patients carry a mutation in the transcription factor II H (TFIIH) subunit TTDA. Using a novel in vivo tripartite split-GFP system, we show that TTDA interacts with the TFIIH subunit p52 and the p52-TTDA-GFP product is incorporated into TFIIH. p52-TTDA-GFP is able to bind DNA and is recruited to UV-damaged DNA. Furthermore, we show that two patient-mutated TTDA proteins can interact with p52, are able to bind to the DNA and can localize to damaged DNA. Our findings give new insights into the behavior of TTDA within the context of a living cell and thereby shed light on the complex phenotype of TTD-A patients.
