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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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Catalytic activity of OGG1–GFP is not required for the recruitment to the chromatin fraction after KBrO3. HeLa cell lines stably expressing OGG1–GFP or the mutant version OGG1(K249Q)–GFP were treated with KBrO3, allowed to recover for 3 h and CSK pre-extracted prior to fixation (A) Heterochromatin is stained with an anti-HP1α (red). Line scans used for plot profiles are indicated in merged images. Scale bar, 2 µm. (B) Repair kinetics of 8-oxoG lesions in OGG1(K249Q)–GFP and OGG1–GFP cells lines by alkaline elution after a 20 mM KBrO3 treatment. (C) Western blot using an anti-GFP antibody showing the recruitment kinetics of OGG1(K249Q)–GFP (left panel) and OGG1–GFP protein (right panel) to the chromatin fraction.
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Figure 7: Catalytic activity of OGG1–GFP is not required for the recruitment to the chromatin fraction after KBrO3. HeLa cell lines stably expressing OGG1–GFP or the mutant version OGG1(K249Q)–GFP were treated with KBrO3, allowed to recover for 3 h and CSK pre-extracted prior to fixation (A) Heterochromatin is stained with an anti-HP1α (red). Line scans used for plot profiles are indicated in merged images. Scale bar, 2 µm. (B) Repair kinetics of 8-oxoG lesions in OGG1(K249Q)–GFP and OGG1–GFP cells lines by alkaline elution after a 20 mM KBrO3 treatment. (C) Western blot using an anti-GFP antibody showing the recruitment kinetics of OGG1(K249Q)–GFP (left panel) and OGG1–GFP protein (right panel) to the chromatin fraction.

Mentions: The correlation between the presence of OGG1 in euchromatin regions and the repair kinetics of 8-oxoG suggests that once repair is accomplished, the DNA glycosylase is released back to the soluble pool. We therefore hypothesized that an OGG1 mutant (K249Q) that recognizes the lesion but is unable to proceed to the excision of the modified base (40) would be retained at the chromatin level. We first confirmed by confocal microscopy that 3 h after KBrO3 treatment OGG1–K249Q was recruited to euchromatin regions in the same way as the wild-type protein (Figure 7A). We then analysed the repair and recruitment kinetics for both forms of the protein. As expected, cells expressing the inactive OGG1–K249Q–GFP were much slower to repair Fpg-sensitive lesions when compared to those expressing the wild-type fusion protein (Figure 7B). Consistent with the notion that repair of the lesion is required to release OGG1 from euchromatin, and in agreement with the presence of un-repaired lesions, western blot analysis of the P1 fractions (Figure 7C) showed that OGG1–K249Q remained tightly associated with the chromatin fraction even 10 h after the end of the treatment, thus supporting the idea that the removal of the oxidized base is required to release the DNA glycosylase from the insoluble fraction associated with euchromatin.Figure 7.


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)

Catalytic activity of OGG1–GFP is not required for the recruitment to the chromatin fraction after KBrO3. HeLa cell lines stably expressing OGG1–GFP or the mutant version OGG1(K249Q)–GFP were treated with KBrO3, allowed to recover for 3 h and CSK pre-extracted prior to fixation (A) Heterochromatin is stained with an anti-HP1α (red). Line scans used for plot profiles are indicated in merged images. Scale bar, 2 µm. (B) Repair kinetics of 8-oxoG lesions in OGG1(K249Q)–GFP and OGG1–GFP cells lines by alkaline elution after a 20 mM KBrO3 treatment. (C) Western blot using an anti-GFP antibody showing the recruitment kinetics of OGG1(K249Q)–GFP (left panel) and OGG1–GFP protein (right panel) to the chromatin fraction.
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Figure 7: Catalytic activity of OGG1–GFP is not required for the recruitment to the chromatin fraction after KBrO3. HeLa cell lines stably expressing OGG1–GFP or the mutant version OGG1(K249Q)–GFP were treated with KBrO3, allowed to recover for 3 h and CSK pre-extracted prior to fixation (A) Heterochromatin is stained with an anti-HP1α (red). Line scans used for plot profiles are indicated in merged images. Scale bar, 2 µm. (B) Repair kinetics of 8-oxoG lesions in OGG1(K249Q)–GFP and OGG1–GFP cells lines by alkaline elution after a 20 mM KBrO3 treatment. (C) Western blot using an anti-GFP antibody showing the recruitment kinetics of OGG1(K249Q)–GFP (left panel) and OGG1–GFP protein (right panel) to the chromatin fraction.
Mentions: The correlation between the presence of OGG1 in euchromatin regions and the repair kinetics of 8-oxoG suggests that once repair is accomplished, the DNA glycosylase is released back to the soluble pool. We therefore hypothesized that an OGG1 mutant (K249Q) that recognizes the lesion but is unable to proceed to the excision of the modified base (40) would be retained at the chromatin level. We first confirmed by confocal microscopy that 3 h after KBrO3 treatment OGG1–K249Q was recruited to euchromatin regions in the same way as the wild-type protein (Figure 7A). We then analysed the repair and recruitment kinetics for both forms of the protein. As expected, cells expressing the inactive OGG1–K249Q–GFP were much slower to repair Fpg-sensitive lesions when compared to those expressing the wild-type fusion protein (Figure 7B). Consistent with the notion that repair of the lesion is required to release OGG1 from euchromatin, and in agreement with the presence of un-repaired lesions, western blot analysis of the P1 fractions (Figure 7C) showed that OGG1–K249Q remained tightly associated with the chromatin fraction even 10 h after the end of the treatment, thus supporting the idea that the removal of the oxidized base is required to release the DNA glycosylase from the insoluble fraction associated with euchromatin.Figure 7.

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