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Assays for chromatin remodeling during nucleotide excision repair in Saccharomyces cerevisiae.

ABSTRACT: How DNA repair proteins interact with the dynamic structure of chromatin is an emerging question. Chromatin structure impedes the access of repair proteins to sites of DNA damage. Several recent studies have implicated chromatin remodeling complexes in DNA repair. In this report we summarize the methods we used to investigate chromatin remodeling during nucleotide excision repair (NER) in vivo. We describe a procedure to analyze UV-induced chromatin remodeling at the silent mating-type locus HML using isolated nuclei from UV-treated yeast cells. In addition, a method to capture transient protein-protein associations in chromatin is outlined. We have used the methods described here to demonstrate that the SWI/SNF chromatin remodeling complex is involved in chromatin rearrangement during NER.

SUBMITTER: Zhang L

PROVIDER: S-EPMC2692424 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Assays for chromatin remodeling during nucleotide excision repair in Saccharomyces cerevisiae.

Zhang Ling L Jones Kristi K Smerdon Michael J MJ Gong Feng F

Methods (San Diego, Calif.) 20090329 1

How DNA repair proteins interact with the dynamic structure of chromatin is an emerging question. Chromatin structure impedes the access of repair proteins to sites of DNA damage. Several recent studies have implicated chromatin remodeling complexes in DNA repair. In this report we summarize the methods we used to investigate chromatin remodeling during nucleotide excision repair (NER) in vivo. We describe a procedure to analyze UV-induced chromatin remodeling at the silent mating-type locus HML ...[more]

PMID: 19336254

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Project description:Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation-induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin.

Dataset Information

Assays for chromatin remodeling during nucleotide excision repair in Saccharomyces cerevisiae.

Publications

Assays for chromatin remodeling during nucleotide excision repair in Saccharomyces cerevisiae.

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