title: 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 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, lambda exonuclease and T4 polymerase 3'-->5'exonuclease, covers approximately 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 approximately 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
authors.forename: C P
authors.initials: CP
authors.lastname: Selby
authors.forename: R
authors.initials: R
authors.lastname: Drapkin
authors.forename: D
authors.initials: D
authors.lastname: Reinberg
authors.forename: A
authors.initials: A
authors.lastname: Sancar
mesh_terms: D002474:Cell-Free System; D018983:DNA Footprinting; D004260:DNA Repair; D006801:Humans; D009690:Nucleic Acid Conformation; D011740:Pyrimidine Dimers / Q000235:genetics*; D012319:RNA Polymerase II / Q000235:genetics*; D013942:Thymine Nucleotides / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D011740:Pyrimidine Dimers; D013942:Thymine Nucleotides; D012319:RNA Polymerase II