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Título

Methods to study transcription-coupled repair in chromatin

AutorGaillard, Hélène ; Wellinger, Ralf Erik ; Aguilera, Andrés
Palabras claveDNA Damage
UV
Cyclobutane pyrimidine dimer
Nucleotide excision repair
Transcription-coupled repair
Chromatin immunoprecipitation
Fecha de publicación2009
EditorHumana Press
CitaciónMethods in molecular biology 523: 141-159 (2009)
ResumenTranscription-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.
DescripciónMethods in Molecular Biology. Chromatin Protocols .-2nd ed. /Edited by Srikumar P. Chellappan. (Series (Springer), 7651)
URIhttp://hdl.handle.net/10261/52497
DOI10.1007/978-1-59745-190-1_10
ISBN978-1-59745-190-1 (e-ISBN)
E-ISSN1940-6029
Identificadoresdoi: 10.1007/978-1-59745-190-1_10
issn: 1064-3745
isbn: 978-1-58829-873-7
ReferenciasPMID: 19381941
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