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Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions.

Amouroux R, Campalans A, Epe B, Radicella JP - Nucleic Acids Res. (2010)

Bottom Line: Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway.We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin.We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.

View Article: PubMed Central - PubMed

Affiliation: CEA, Institut de Radiobiologie Cellulaire et Moléculaire, 18 route du Panorama, UMR217 F-92265 Fontenay aux Roses, France.

ABSTRACT
How DNA repair machineries detect and access, within the context of chromatin, lesions inducing little or no distortion of the DNA structure is a poorly understood process. Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway. We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin. The underlying mechanism does not require direct interaction of the protein with the oxidized base, however, the release of the protein from the chromatin fraction requires completion of repair. Inducing chromatin compaction by sucrose results in a complete but reversible inhibition of the in vivo repair of 8-oxoguanine. We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.

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Overexpression of OGG1–GFP accelerates repair of 8-oxoG induced in KBrO3-treated cells. (A) 8-OxoG were stained with anti-8-oxoG antibody; DNA was stained with PI after RNAse A digestion. Scale bars, 2 µm. (B) 8-OxoG in untreated and KBrO3-treated cells were quantified as Fpg-sensitive sites by alkaline elution in HeLa and OGG1–GFP cells. (C) Kinetics of repair of 8-oxoG in HeLa and OGG1–GFP cells. After KBrO3 treatments cells were allowed to recover for the indicated periods of time and analysed by alkaline elution to measure the remaining 8-oxoG lesions.
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Figure 1: Overexpression of OGG1–GFP accelerates repair of 8-oxoG induced in KBrO3-treated cells. (A) 8-OxoG were stained with anti-8-oxoG antibody; DNA was stained with PI after RNAse A digestion. Scale bars, 2 µm. (B) 8-OxoG in untreated and KBrO3-treated cells were quantified as Fpg-sensitive sites by alkaline elution in HeLa and OGG1–GFP cells. (C) Kinetics of repair of 8-oxoG in HeLa and OGG1–GFP cells. After KBrO3 treatments cells were allowed to recover for the indicated periods of time and analysed by alkaline elution to measure the remaining 8-oxoG lesions.

Mentions: To explore the cellular DNA repair mechanisms required for the removal of 8-oxoG from chromosomal DNA we used KBrO3, a carcinogenic agent known to induce oxidative stress in eukaryotic cells. Genotoxicity requires reduction of bromate by thiols (as glutathione or reduced cysteines) and induces predominantly 8-oxoG lesions in DNA (37). As displayed in Figure 1A, immediately after a 30 min treatment with 40 mM KBrO3, HeLa cells immunostained with an antibody against 8-oxoG showed a strong fluorescent signal compared to NT cells. Fpg-sensitive lesions, mostly 8-oxoG, were quantified by alkaline elution. The steady-state level of endogenous Fpg-sensitive sites was ∼0.21 lesions/106 bp in both HeLa and HeLa cells expressing an OGG1–GFP fusion protein. KBrO3 treatment induced a 10-fold increase in Fpg-sensitive lesions (3.33 and 2.36 lesions/106 bp in HeLa and OGG1–GFP cells, respectively; Figure 1B). Because extracts from cells expressing the OGG1 fusion have about 10-fold more 8-oxoG DNA glycosylase activity, the difference in the number of induced lesions is probably due to a beginning of repair during the 30 min treatment. The DNA glycosylase activity is not affected by the exposure to KBrO3 (Supplementary Figure S1).Figure 1.


Oxidative stress triggers the preferential assembly of base excision repair complexes on open chromatin regions.

Amouroux R, Campalans A, Epe B, Radicella JP - Nucleic Acids Res. (2010)

Overexpression of OGG1–GFP accelerates repair of 8-oxoG induced in KBrO3-treated cells. (A) 8-OxoG were stained with anti-8-oxoG antibody; DNA was stained with PI after RNAse A digestion. Scale bars, 2 µm. (B) 8-OxoG in untreated and KBrO3-treated cells were quantified as Fpg-sensitive sites by alkaline elution in HeLa and OGG1–GFP cells. (C) Kinetics of repair of 8-oxoG in HeLa and OGG1–GFP cells. After KBrO3 treatments cells were allowed to recover for the indicated periods of time and analysed by alkaline elution to measure the remaining 8-oxoG lesions.
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Figure 1: Overexpression of OGG1–GFP accelerates repair of 8-oxoG induced in KBrO3-treated cells. (A) 8-OxoG were stained with anti-8-oxoG antibody; DNA was stained with PI after RNAse A digestion. Scale bars, 2 µm. (B) 8-OxoG in untreated and KBrO3-treated cells were quantified as Fpg-sensitive sites by alkaline elution in HeLa and OGG1–GFP cells. (C) Kinetics of repair of 8-oxoG in HeLa and OGG1–GFP cells. After KBrO3 treatments cells were allowed to recover for the indicated periods of time and analysed by alkaline elution to measure the remaining 8-oxoG lesions.
Mentions: To explore the cellular DNA repair mechanisms required for the removal of 8-oxoG from chromosomal DNA we used KBrO3, a carcinogenic agent known to induce oxidative stress in eukaryotic cells. Genotoxicity requires reduction of bromate by thiols (as glutathione or reduced cysteines) and induces predominantly 8-oxoG lesions in DNA (37). As displayed in Figure 1A, immediately after a 30 min treatment with 40 mM KBrO3, HeLa cells immunostained with an antibody against 8-oxoG showed a strong fluorescent signal compared to NT cells. Fpg-sensitive lesions, mostly 8-oxoG, were quantified by alkaline elution. The steady-state level of endogenous Fpg-sensitive sites was ∼0.21 lesions/106 bp in both HeLa and HeLa cells expressing an OGG1–GFP fusion protein. KBrO3 treatment induced a 10-fold increase in Fpg-sensitive lesions (3.33 and 2.36 lesions/106 bp in HeLa and OGG1–GFP cells, respectively; Figure 1B). Because extracts from cells expressing the OGG1 fusion have about 10-fold more 8-oxoG DNA glycosylase activity, the difference in the number of induced lesions is probably due to a beginning of repair during the 30 min treatment. The DNA glycosylase activity is not affected by the exposure to KBrO3 (Supplementary Figure S1).Figure 1.

Bottom Line: Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway.We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin.We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.

View Article: PubMed Central - PubMed

Affiliation: CEA, Institut de Radiobiologie Cellulaire et Moléculaire, 18 route du Panorama, UMR217 F-92265 Fontenay aux Roses, France.

ABSTRACT
How DNA repair machineries detect and access, within the context of chromatin, lesions inducing little or no distortion of the DNA structure is a poorly understood process. Removal of oxidized bases is initiated by a DNA glycosylase that recognises and excises the damaged base, initiating the base excision repair (BER) pathway. We show that upon induction of 8-oxoguanine, a mutagenic product of guanine oxidation, the mammalian 8-oxoguanine DNA glycosylase OGG1 is recruited together with other proteins involved in BER to euchromatin regions rich in RNA and RNA polymerase II and completely excluded from heterochromatin. The underlying mechanism does not require direct interaction of the protein with the oxidized base, however, the release of the protein from the chromatin fraction requires completion of repair. Inducing chromatin compaction by sucrose results in a complete but reversible inhibition of the in vivo repair of 8-oxoguanine. We conclude that after induction of oxidative DNA damage, the DNA glycosylase is actively recruited to regions of open chromatin allowing the access of the BER machinery to the lesions, suggesting preferential repair of active chromosome regions.

Show MeSH
Related in: MedlinePlus