RNA polymerase II stalled at a thymine dimer: footprint and effect on excision repair

• Published in: Nucleic acids research 1997-02, Vol.25 (4), p.787-793
• Main Authors: Selby, Christopher P., Drapkin, Ronny, Reinberg, Danny, Sancar, Aziz
• Format: Article
• Language: English
• Subjects: Adenovirus; Cell-Free System; DNA Footprinting; DNA Repair; Humans; Nucleic Acid Conformation; Pyrimidine Dimers - genetics; RNA Polymerase II - genetics; Thymine Nucleotides - genetics; Transcription, Genetic
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/25.4.787

Bulky lesions in the template strand block the progression of RNA polymerase II (RNAP II) and are repaired more rapidly than lesions in the non-transcribed strand, which do not block transcription. In order to better understand the basis of this transcription-coupled repair we developed an in vitro system with purified transcription and nucleotide excision repair proteins and a plasmid containing the adenovirus major late promoter and a thymine dimer in the template strand downstream of the transcription start site. The footprint of RNAP II stalled at the thymine dimer, obtained using DNase I, λ exonuclease and T4 polymerase 3′→5′ exonuclease, covers ∼40 nt and is nearly symmetrical around the dimer. The ternary complex formed at the lesion site is rather stable, with a half-life of ∼20 h. Surprisingly, addition of human repair proteins results in repair of transcription-blocking dimers in the ternary complex. The blocked polymerase neither inhibits nor stimulates repair and repair is observed in the absence of CSB protein, the putative human transcription—repair coupling factor.