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

Abstract

Bulky lesions in the template strand block the progression of RNA polymerase H (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.