Methods to study transcription-coupled repair in chromatin

Methods Mol Biol. 2009:523:141-59. doi: 10.1007/978-1-59745-190-1_10.

Abstract

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair that allows for the enhanced repair of the transcribed strand of active genes. A classical method to study DNA repair in vivo consists in the molecular analysis of UV-induced DNA damages at specific loci. Cells are irradiated with a defined dose of UV light leading to the formation of DNA lesions and incubated in the dark to allow repair. About 90% of the photoproducts consist of cyclobutane pyrimidine dimers, which can be cleaved by the DNA nicking activity of the T4 endonuclease V (T4endoV) repair enzyme. Strand-specific repair in a suitable restriction fragment is determined by alkaline gel electrophoresis followed by Southern blot transfer and indirect end-labeling using a single-stranded probe. Recent approaches have assessed the role of transcription factors in TCR by analyzing RNA polymerase II occupancy on a damaged template by chromatin immunoprecipitation (ChIP). Cells are treated with formaldehyde in vivo to cross-link proteins to DNA and enrichment of a protein of interest is done by subsequent immunoprecipitation. Upon reversal of the protein-DNA cross-links, the amount of coprecipitated DNA fragments can be detected by quantitative PCR. To perform ChIP on UV-damaged templates, we included an in vitro photoreactivation step prior to PCR analysis to ensure that all precipitated DNA fragments serve as substrates for the PCR reaction. Here, we provide a detailed protocol for both the DNA repair analysis and the ChIP approaches to study TCR in chromatin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / metabolism*
  • Chromatin Immunoprecipitation
  • DNA Repair* / radiation effects
  • DNA, Fungal / isolation & purification
  • Kinetics
  • Molecular Biology / methods*
  • Pyrimidine Dimers / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae / radiation effects
  • Transcription, Genetic* / radiation effects
  • Ultraviolet Rays

Substances

  • Chromatin
  • DNA, Fungal
  • Pyrimidine Dimers