Limits...
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.

Show MeSH

Related in: MedlinePlus

Chromatin from a or a mating type cells was incubated with Mnase and processed as in [15]. Scans are shown of band intensities on agarose gels; the higher the peaks then the more efficient the cutting by Mnase. The absence or reduction in peaks in α cells as opposed to in a cells relates to the positioning of 4 nucleosomes as indicated; each occupies about 147 bp of the MFA2 sequence shown underneath.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-ijms-13-11141: Chromatin from a or a mating type cells was incubated with Mnase and processed as in [15]. Scans are shown of band intensities on agarose gels; the higher the peaks then the more efficient the cutting by Mnase. The absence or reduction in peaks in α cells as opposed to in a cells relates to the positioning of 4 nucleosomes as indicated; each occupies about 147 bp of the MFA2 sequence shown underneath.

Mentions: Hence we were in a position whereby we could examine the nuances of GG-NER at nucleotide resolution in genes of choice. However, we wished to relate these events to changes in chromatin that were linked to enabling efficient GG-NER. Therefore we needed to examine the nature of any nucleosomes at MFA2 in both a and α cells so as we could relate our repair data to the presence of nucleosomes, their positions, or their absence and their modifications. We reasoned that we could adapt our method to detect CPDs at nucleotide resolution in order to determine nucleosome positions at MFA2. This was undertaken not by examining the results of cutting with a CPD specific enzyme as for DNA damage analyses, but by examining the results of cutting DNA in chromatin with Mnase; a standard way of determining nucleosome positions at low resolution [38]. To afford a high resolution analysis we then purified DNA fragments of choice exactly as we did for the same fragments when analysing DNA damage. We then radiolabelled and ran those fragments on alkaline agarose gels to identify any MNase protected regions at MFA2. If protected regions equivalent to lengths of about 147 base pairs are identified then this is an indication of the presence of a nucleosome in that region [15]. As shown in Figure 5 there were no fixed nucleosomes in chromatin from a mating type cells where MFA2 is transcriptionally active but in α mating type cells where the gene is repressed there are four positioned nucleosomes in the chromatin. Two of these nucleosomes reside in the MFA2 regulatory region, either side of the Mcm1 binding site with that nucleosome labelled as −1 being over the TATA box. Then there are 2 positioned nucleosomes within the MFA2 coding region of this relatively small gene.


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)

Chromatin from a or a mating type cells was incubated with Mnase and processed as in [15]. Scans are shown of band intensities on agarose gels; the higher the peaks then the more efficient the cutting by Mnase. The absence or reduction in peaks in α cells as opposed to in a cells relates to the positioning of 4 nucleosomes as indicated; each occupies about 147 bp of the MFA2 sequence shown underneath.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-ijms-13-11141: Chromatin from a or a mating type cells was incubated with Mnase and processed as in [15]. Scans are shown of band intensities on agarose gels; the higher the peaks then the more efficient the cutting by Mnase. The absence or reduction in peaks in α cells as opposed to in a cells relates to the positioning of 4 nucleosomes as indicated; each occupies about 147 bp of the MFA2 sequence shown underneath.
Mentions: Hence we were in a position whereby we could examine the nuances of GG-NER at nucleotide resolution in genes of choice. However, we wished to relate these events to changes in chromatin that were linked to enabling efficient GG-NER. Therefore we needed to examine the nature of any nucleosomes at MFA2 in both a and α cells so as we could relate our repair data to the presence of nucleosomes, their positions, or their absence and their modifications. We reasoned that we could adapt our method to detect CPDs at nucleotide resolution in order to determine nucleosome positions at MFA2. This was undertaken not by examining the results of cutting with a CPD specific enzyme as for DNA damage analyses, but by examining the results of cutting DNA in chromatin with Mnase; a standard way of determining nucleosome positions at low resolution [38]. To afford a high resolution analysis we then purified DNA fragments of choice exactly as we did for the same fragments when analysing DNA damage. We then radiolabelled and ran those fragments on alkaline agarose gels to identify any MNase protected regions at MFA2. If protected regions equivalent to lengths of about 147 base pairs are identified then this is an indication of the presence of a nucleosome in that region [15]. As shown in Figure 5 there were no fixed nucleosomes in chromatin from a mating type cells where MFA2 is transcriptionally active but in α mating type cells where the gene is repressed there are four positioned nucleosomes in the chromatin. Two of these nucleosomes reside in the MFA2 regulatory region, either side of the Mcm1 binding site with that nucleosome labelled as −1 being over the TATA box. Then there are 2 positioned nucleosomes within the MFA2 coding region of this relatively small gene.

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