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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

Exponential phase a mating type cells either with or without Gcn5 were UV irradiated with 70 J/m2 of UV at 260 nm and either sampled immediately or after 1, 2 or 3 h post UV in YC medium for nucleotide excision repair (NER) to occur. DNA was purified and incubated with (+) or without (−) a CPD specific DNA glyscosylase. The DNA was run on alkaline agarose gels before Southern blotting to analyse the non-transcribed strand and hence the frequency of GG-NER. Loss of signal is indicative of CPDs and the return of signal indicative of their repair. The return of signals is plotted graphically as % repair [11].
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f3-ijms-13-11141: Exponential phase a mating type cells either with or without Gcn5 were UV irradiated with 70 J/m2 of UV at 260 nm and either sampled immediately or after 1, 2 or 3 h post UV in YC medium for nucleotide excision repair (NER) to occur. DNA was purified and incubated with (+) or without (−) a CPD specific DNA glyscosylase. The DNA was run on alkaline agarose gels before Southern blotting to analyse the non-transcribed strand and hence the frequency of GG-NER. Loss of signal is indicative of CPDs and the return of signal indicative of their repair. The return of signals is plotted graphically as % repair [11].

Mentions: Figure 2 shows slot blots of CPD incidence at various times after UV exposure in cells with or without Gcn5. There is no discernable differences in NER from the genome overall. However, when we examined the changes in CPD frequency in MFA2 versus the RBP2 genes there was much less efficient NER at the MFA2 gene in the absence of Gcn5 but no significant change in NER at RPB2. Figure 3 shows Southern blots to detect CPDs in the MFA2 and RPB2 genes. There is a clear and statistically significant defect in NER at MFA2 but not at RPB2. These data show that NER is indeed influenced, but only at some locations within the genome.


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)

Exponential phase a mating type cells either with or without Gcn5 were UV irradiated with 70 J/m2 of UV at 260 nm and either sampled immediately or after 1, 2 or 3 h post UV in YC medium for nucleotide excision repair (NER) to occur. DNA was purified and incubated with (+) or without (−) a CPD specific DNA glyscosylase. The DNA was run on alkaline agarose gels before Southern blotting to analyse the non-transcribed strand and hence the frequency of GG-NER. Loss of signal is indicative of CPDs and the return of signal indicative of their repair. The return of signals is plotted graphically as % repair [11].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijms-13-11141: Exponential phase a mating type cells either with or without Gcn5 were UV irradiated with 70 J/m2 of UV at 260 nm and either sampled immediately or after 1, 2 or 3 h post UV in YC medium for nucleotide excision repair (NER) to occur. DNA was purified and incubated with (+) or without (−) a CPD specific DNA glyscosylase. The DNA was run on alkaline agarose gels before Southern blotting to analyse the non-transcribed strand and hence the frequency of GG-NER. Loss of signal is indicative of CPDs and the return of signal indicative of their repair. The return of signals is plotted graphically as % repair [11].
Mentions: Figure 2 shows slot blots of CPD incidence at various times after UV exposure in cells with or without Gcn5. There is no discernable differences in NER from the genome overall. However, when we examined the changes in CPD frequency in MFA2 versus the RBP2 genes there was much less efficient NER at the MFA2 gene in the absence of Gcn5 but no significant change in NER at RPB2. Figure 3 shows Southern blots to detect CPDs in the MFA2 and RPB2 genes. There is a clear and statistically significant defect in NER at MFA2 but not at RPB2. These data show that NER is indeed influenced, but only at some locations within the genome.

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