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dc.contributor.author | Gaillard, Hélène | - |
dc.contributor.author | Wellinger, Ralf Erik | - |
dc.contributor.author | Aguilera, Andrés | - |
dc.date.accessioned | 2012-06-28T11:59:30Z | - |
dc.date.available | 2012-06-28T11:59:30Z | - |
dc.date.issued | 2009 | - |
dc.identifier | doi: 10.1007/978-1-59745-190-1_10 | - |
dc.identifier | issn: 1064-3745 | - |
dc.identifier | isbn: 978-1-58829-873-7 | - |
dc.identifier.citation | Methods in molecular biology 523: 141-159 (2009) | - |
dc.identifier.isbn | 978-1-59745-190-1 (e-ISBN) | - |
dc.identifier.other | PMID: 19381941 | - |
dc.identifier.uri | http://hdl.handle.net/10261/52497 | - |
dc.description | Methods in Molecular Biology. Chromatin Protocols .-2nd ed. /Edited by Srikumar P. Chellappan. (Series (Springer), 7651) | - |
dc.description.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. | - |
dc.description.sponsorship | Research in AA’s laboratory is funded by grants from the Spanish Ministery of Science and Innovation and from the Junta de Andalucia. | - |
dc.language.iso | eng | - |
dc.publisher | Humana Press | - |
dc.rights | closedAccess | - |
dc.subject | DNA damage | - |
dc.subject | UV | - |
dc.subject | Cyclobutane pyrimidine dimers | - |
dc.subject | Nucleotide excision repair | - |
dc.subject | Transcription-coupled repair | - |
dc.subject | Chromatin immunoprecipitation | - |
dc.title | Methods to study transcription-coupled repair in chromatin | - |
dc.type | capítulo de libro | - |
dc.identifier.doi | 10.1007/978-1-59745-190-1_10 | - |
dc.identifier.e-issn | 1940-6029 | - |
dc.date.updated | 2012-06-28T11:59:30Z | - |
dc.description.version | Peer Reviewed | - |
dc.type.coar | http://purl.org/coar/resource_type/c_3248 | es_ES |
item.grantfulltext | none | - |
item.cerifentitytype | Publications | - |
item.openairecristype | http://purl.org/coar/resource_type/c_18cf | - |
item.languageiso639-1 | en | - |
item.fulltext | No Fulltext | - |
item.openairetype | capítulo de libro | - |
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