RNA Polymerase II Stalled at a Thymine Dimer: Footprint and Effect on Excision Repair

Author: Selby Christopher P.   Reinberg Danny   Sancar Aziz  

Publisher: Oxford University Press

ISSN: 1362-4962

Source: Nucleic Acids Research, Vol.25, Iss.4, 1997-01, pp. : 787-793

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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, 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.

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