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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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(A) The domain structure of Rad16; (B) UV survival curves of the strains indicated; (C) Histone H3 acetylation at the MFA2 promoter. ChIP analysis of Histone H3 acetylation (H3Ac) was performed using H3Ac (Lys 9 and Lys 14) antibody. U: untreated samples; 0: cells received 100 J/m2 of ultraviolet without repair; 15 and 60: cells were irradiated with UV and then were allowed to repair in medium for the times indicated. Acetylation level shown as the fold change relative to unirradiated cells; (D) The occupancy of Gcn5 at the MFA2 promoter. ChIP was performed with anti-myc antibody. Gcn5 binding is presented as the fold change relative to untreated cells. [20].
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f12-ijms-13-11141: (A) The domain structure of Rad16; (B) UV survival curves of the strains indicated; (C) Histone H3 acetylation at the MFA2 promoter. ChIP analysis of Histone H3 acetylation (H3Ac) was performed using H3Ac (Lys 9 and Lys 14) antibody. U: untreated samples; 0: cells received 100 J/m2 of ultraviolet without repair; 15 and 60: cells were irradiated with UV and then were allowed to repair in medium for the times indicated. Acetylation level shown as the fold change relative to unirradiated cells; (D) The occupancy of Gcn5 at the MFA2 promoter. ChIP was performed with anti-myc antibody. Gcn5 binding is presented as the fold change relative to untreated cells. [20].

Mentions: To examine the role of Rad16 further we decided to create mutants at each of its two active sites, namely the ATPase domain and the site for E3 ubiquitin ligase activity associated with the C3HC4 RING domain that is embedded within the ATPase domain. We mutated either each one alone or both of these in combination [20]. Creation of the point mutations within the Rad16 ATPase domain (K216A), RING-finger domain (C552A,H554A) and double mutation of ATPase and RING-finger domains (K216A,C552A,H554A), resulting in the amino-acid substitutions, lysine 216 to alanine, cysteine 552 to alanine and histidine 554 to alanine. This approach revealed that mutations in both its ATPase site and its ring domain were required to totally inactivate the recruitment of Gcn5 and the subsequent Histone H3 acetylation at lysines 9/14 (Figure 12). Figure 13 shows how the repair of CPDs within the MFA2 regulatory region is reduced in the ATPase and ring domain double mutant (A) and how this impinges on the accessibility of the RsaI site residing within the core of nucleosome −2 (B).


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) The domain structure of Rad16; (B) UV survival curves of the strains indicated; (C) Histone H3 acetylation at the MFA2 promoter. ChIP analysis of Histone H3 acetylation (H3Ac) was performed using H3Ac (Lys 9 and Lys 14) antibody. U: untreated samples; 0: cells received 100 J/m2 of ultraviolet without repair; 15 and 60: cells were irradiated with UV and then were allowed to repair in medium for the times indicated. Acetylation level shown as the fold change relative to unirradiated cells; (D) The occupancy of Gcn5 at the MFA2 promoter. ChIP was performed with anti-myc antibody. Gcn5 binding is presented as the fold change relative to untreated cells. [20].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f12-ijms-13-11141: (A) The domain structure of Rad16; (B) UV survival curves of the strains indicated; (C) Histone H3 acetylation at the MFA2 promoter. ChIP analysis of Histone H3 acetylation (H3Ac) was performed using H3Ac (Lys 9 and Lys 14) antibody. U: untreated samples; 0: cells received 100 J/m2 of ultraviolet without repair; 15 and 60: cells were irradiated with UV and then were allowed to repair in medium for the times indicated. Acetylation level shown as the fold change relative to unirradiated cells; (D) The occupancy of Gcn5 at the MFA2 promoter. ChIP was performed with anti-myc antibody. Gcn5 binding is presented as the fold change relative to untreated cells. [20].
Mentions: To examine the role of Rad16 further we decided to create mutants at each of its two active sites, namely the ATPase domain and the site for E3 ubiquitin ligase activity associated with the C3HC4 RING domain that is embedded within the ATPase domain. We mutated either each one alone or both of these in combination [20]. Creation of the point mutations within the Rad16 ATPase domain (K216A), RING-finger domain (C552A,H554A) and double mutation of ATPase and RING-finger domains (K216A,C552A,H554A), resulting in the amino-acid substitutions, lysine 216 to alanine, cysteine 552 to alanine and histidine 554 to alanine. This approach revealed that mutations in both its ATPase site and its ring domain were required to totally inactivate the recruitment of Gcn5 and the subsequent Histone H3 acetylation at lysines 9/14 (Figure 12). Figure 13 shows how the repair of CPDs within the MFA2 regulatory region is reduced in the ATPase and ring domain double mutant (A) and how this impinges on the accessibility of the RsaI site residing within the core of nucleosome −2 (B).

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