Methods to study transcription-coupled repair in chromatin.
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.
Gaillard
Hélène
H
CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain.
Wellinger
Ralf Erik
RE
Aguilera
Andrés
A
D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004260:DNA Repair / Q000528:radiation effects; D004271:DNA, Fungal / Q000302:isolation & purification; D007700:Kinetics; D008967:Molecular Biology / Q000379:methods*; D011740:Pyrimidine Dimers / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000528:radiation effects; D014158:Transcription, Genetic / Q000528:radiation effects; D014466:Ultraviolet Rays
D002843:Chromatin; D004271:DNA, Fungal; D011740:Pyrimidine Dimers