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Nucleotide excision repair in cellular chromatin: studies with yeast from nucleotide to gene to genome.

Waters R, Evans K, Bennett M, Yu S, Reed S - Int J Mol Sci (2012)

Bottom Line: Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae.We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences.In the latter case we also see a role for acetylation at histone H4.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK; E-Mails: evansKE3@cardiff.ac.uk (K.E.); bennettMR1@cardiff.ac.uk (M.B.); yuS@cardiff.ac.uk (S.Y.); reedSH1@cardiff.ac.uk (S.R.).

ABSTRACT
Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

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Related in: MedlinePlus

A restriction enzyme accessibility assay to examine the relationship between nucleosomes and core DNA. Chromatin was digested with either Restriction Enzyme I (HaeIII) alone or with HaeIII plus Enzyme II (RsaI). HaeIII sites occur either side of the −2 positioned nucleosome of the repressed MFA2 gene, whereas the RsaI site resides within the core DNA of this nucleosome. DNA from these digests was run on agarose gels and probed with a radiolabelled sequence as indicated and described in [16].
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f7-ijms-13-11141: A restriction enzyme accessibility assay to examine the relationship between nucleosomes and core DNA. Chromatin was digested with either Restriction Enzyme I (HaeIII) alone or with HaeIII plus Enzyme II (RsaI). HaeIII sites occur either side of the −2 positioned nucleosome of the repressed MFA2 gene, whereas the RsaI site resides within the core DNA of this nucleosome. DNA from these digests was run on agarose gels and probed with a radiolabelled sequence as indicated and described in [16].

Mentions: Hence Gcn5 had a role in facilitating efficient GG-NER at MFA2. This result is not restricted to MFA2 and we also observed the same effects at MET16 when it was transcriptionally repressed or transcriptionally active [18]. Importantly, this role of Gcn5 in GG-NER at MFA2 was totally outside of its role in transcription in a mating type cells because we showed that this H3 acetylation after UV in these cells was not related to any aberrant transcriptional activation of this repressed locus [16]. Unlike in un-irradiated a mating type cells, there was no detectable recruitment of TATA box binding protein to the MFA2 TATA box in UV treated α cells, nor was there any detectable MFA2 transcript in these cells. At the same two fixed nucleosomes of the MFA2 regulatory region in α mating type cells we examined in chromatin the accessibility of restriction sites within the nucleosomal core DNA [16]. Figure 7 describes the logic behind this assay and provides images of gels showing the changes in fragment size in DNA from the chromatin for nucleosome −2. The assay was performed on cells of both mating types and in cells where the Tup1 repressor which represses MFA2 was deleted [38,39]. This deletion de-represses the gene and creates a hyperacetylated histone H3 state at lysines 9/14 in the nucleosomes of the regulatory region. We additionally examined events in TUP1 deleted cells where there was also a mutated TATA box to prevent transcription; so here we had created chromatin that was more accessible and hyperacetylated, but in the absence of MFA2’s transcription and any potential TC-NER.


Nucleotide excision repair in cellular chromatin: studies with yeast from nucleotide to gene to genome.

Waters R, Evans K, Bennett M, Yu S, Reed S - Int J Mol Sci (2012)

A restriction enzyme accessibility assay to examine the relationship between nucleosomes and core DNA. Chromatin was digested with either Restriction Enzyme I (HaeIII) alone or with HaeIII plus Enzyme II (RsaI). HaeIII sites occur either side of the −2 positioned nucleosome of the repressed MFA2 gene, whereas the RsaI site resides within the core DNA of this nucleosome. DNA from these digests was run on agarose gels and probed with a radiolabelled sequence as indicated and described in [16].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472735&req=5

f7-ijms-13-11141: A restriction enzyme accessibility assay to examine the relationship between nucleosomes and core DNA. Chromatin was digested with either Restriction Enzyme I (HaeIII) alone or with HaeIII plus Enzyme II (RsaI). HaeIII sites occur either side of the −2 positioned nucleosome of the repressed MFA2 gene, whereas the RsaI site resides within the core DNA of this nucleosome. DNA from these digests was run on agarose gels and probed with a radiolabelled sequence as indicated and described in [16].
Mentions: Hence Gcn5 had a role in facilitating efficient GG-NER at MFA2. This result is not restricted to MFA2 and we also observed the same effects at MET16 when it was transcriptionally repressed or transcriptionally active [18]. Importantly, this role of Gcn5 in GG-NER at MFA2 was totally outside of its role in transcription in a mating type cells because we showed that this H3 acetylation after UV in these cells was not related to any aberrant transcriptional activation of this repressed locus [16]. Unlike in un-irradiated a mating type cells, there was no detectable recruitment of TATA box binding protein to the MFA2 TATA box in UV treated α cells, nor was there any detectable MFA2 transcript in these cells. At the same two fixed nucleosomes of the MFA2 regulatory region in α mating type cells we examined in chromatin the accessibility of restriction sites within the nucleosomal core DNA [16]. Figure 7 describes the logic behind this assay and provides images of gels showing the changes in fragment size in DNA from the chromatin for nucleosome −2. The assay was performed on cells of both mating types and in cells where the Tup1 repressor which represses MFA2 was deleted [38,39]. This deletion de-represses the gene and creates a hyperacetylated histone H3 state at lysines 9/14 in the nucleosomes of the regulatory region. We additionally examined events in TUP1 deleted cells where there was also a mutated TATA box to prevent transcription; so here we had created chromatin that was more accessible and hyperacetylated, but in the absence of MFA2’s transcription and any potential TC-NER.

Bottom Line: Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae.We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences.In the latter case we also see a role for acetylation at histone H4.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK; E-Mails: evansKE3@cardiff.ac.uk (K.E.); bennettMR1@cardiff.ac.uk (M.B.); yuS@cardiff.ac.uk (S.Y.); reedSH1@cardiff.ac.uk (S.R.).

ABSTRACT
Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

Show MeSH
Related in: MedlinePlus