Fiche publication
Date publication
janvier 2006
Auteurs
Membres identifiés du Cancéropôle Est :
Dr EGLY Jean-Marc
Tous les auteurs :
Laine JP, Mocquet V, Egly JM
Lien Pubmed
Résumé
Transcription and nucleotide excision repair (NER) are two major mechanisms in which the transcription factor TFIIH plays a crucial role. In order to investigate its function, we first described a fast and efficient purification protocol of TFIIH from either HeLa cells or patient cell lines, as well as various in vitro enzymatic assays set up in our laboratory. All these enzymatic assays have been adapted to work on immobilized DNA, a powerful tool allowing for sequential protein incubations in various buffer conditions, without destabilizing protein complexes bound to the DNA. Runoff transcription assays performed with either whole cell extract or highly purified factors underline the role of TFIIH helicases (XPB and XPD) in the RNA synthesis. Moreover, the requirement of XPB and XPD in NER can also be investigated with various assays corresponding to the different steps of this process. The DNA opening assay (permanganate footprint) highlights DNA unwinding of the double-stranded DNA fragment within the repair complex, whereas the dual incision assay allows for detection of the double cut on both sides of the lesion. The gap-filling reaction following the cuts can be monitored as well with a DNA resynthesis assay. Futhermore, the use of immobilized DNA is of great interest to study the detailed mechanism in which TFIIH plays a central role. This chapter describes the ATP-independent recruitment of TFIIH on the damaged DNA previously recognized by XPC-hHR23B and the sequential arrival and departure of the repair proteins within the NER complex.
Référence
Methods Enzymol. 2006;408:246-63.
