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Methods Mol Biol. 2015;1288:273-88. doi: 10.1007/978-1-4939-2474-5_15.

Methods to study transcription-coupled repair in chromatin.

Author information

1
Genetics, CABIMER, Universidad de Sevilla-CSIC, Avenida Américo Vespucio s/n, 41092, Seville, Spain.

Abstract

The effect of endogenous and exogenous DNA damage on the cellular metabolism can be studied at the genetic and molecular level. A paradigmatic case is the repair of UV-induced pyrimidine dimers (PDs) by nucleotide excision repair (NER) in Saccharomyces cerevisiae. To follow the formation and repair of PDs at specific chromosome loci, cells are irradiated with UV-light and incubated in the dark to allow repair by NER. Upon DNA isolation, cyclobutane pyrimidine dimers, which account for about 90 % of PDs, can be cleaved in vitro by the DNA nicking activity of the T4 endonuclease V repair enzyme. Subsequently, strand-specific repair in a suitable restriction fragment is determined by denaturing gel electrophoresis followed by Southern blot and indirect end-labeling using a single-stranded DNA probe. Noteworthy, this protocol could potentially be adapted to other kind of DNA lesions, as long as a DNA nick is formed or a lesion-specific endonuclease is available.Transcription-coupled repair (TC-NER) is a sub-pathway of NER that catalyzes the repair of the transcribed strand of active genes. RNA polymerase II is essential for TC-NER, and its occupancy on a damaged template can be analyzed by chromatin immunoprecipitation (ChIP). In this chapter, we provide an up-dated protocol for both the DNA repair analysis and ChIP approaches to study TC-NER in yeast chromatin.

PMID:
25827885
DOI:
10.1007/978-1-4939-2474-5_15
[Indexed for MEDLINE]

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