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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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Related in: MedlinePlus

After KBrO3 OGG1 is relocalized to foci resistant to detergent-containing buffer. (A) Subcellular fractionation of NT and KBrO3-treated OGG1–GFP cells. Cells were separated into fractions S1 (soluble proteins) and P1 (chromatin fraction) and different fractions analysed by western blot using an anti-GFP antibody. Lamin B1 was used as a loading control. (B) Distribution patterns of OGG1–GFP in NT and KBrO3-treated cells. Prior to fixation, soluble proteins were removed with CSK-0.5% triton when indicated. (C) NT cells expressing OGG1–GFP were directly fixed, DAPI stained and analysed by confocal microscopy. DAPI staining was used to define heterochromatin regions (white outlines). Scale bars, 2 µm.
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Figure 2: After KBrO3 OGG1 is relocalized to foci resistant to detergent-containing buffer. (A) Subcellular fractionation of NT and KBrO3-treated OGG1–GFP cells. Cells were separated into fractions S1 (soluble proteins) and P1 (chromatin fraction) and different fractions analysed by western blot using an anti-GFP antibody. Lamin B1 was used as a loading control. (B) Distribution patterns of OGG1–GFP in NT and KBrO3-treated cells. Prior to fixation, soluble proteins were removed with CSK-0.5% triton when indicated. (C) NT cells expressing OGG1–GFP were directly fixed, DAPI stained and analysed by confocal microscopy. DAPI staining was used to define heterochromatin regions (white outlines). Scale bars, 2 µm.

Mentions: To determine OGG1 localization during the repair period, after the 30 min KBrO3 treatment cells were allowed to recover for 3 h in fresh medium before proceeding to subcellular fractionation. The soluble pool of proteins (S1), corresponding to the cytoplasm and nucleoplasm, was obtained by washing the cells with CSK buffer containing 0.5% triton. The resulting pellet (P1) corresponded to chromatin- and matrix-associated proteins. As revealed by western blot analysis of the S1 fraction, the majority of OGG1 remained soluble after KBrO3. However, whereas no retention of OGG1 could be observed in NT cells, KBrO3 induced the association of a fraction of hOGG1 with chromatin and nuclear matrix (Figure 2A).Figure 2.


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)

After KBrO3 OGG1 is relocalized to foci resistant to detergent-containing buffer. (A) Subcellular fractionation of NT and KBrO3-treated OGG1–GFP cells. Cells were separated into fractions S1 (soluble proteins) and P1 (chromatin fraction) and different fractions analysed by western blot using an anti-GFP antibody. Lamin B1 was used as a loading control. (B) Distribution patterns of OGG1–GFP in NT and KBrO3-treated cells. Prior to fixation, soluble proteins were removed with CSK-0.5% triton when indicated. (C) NT cells expressing OGG1–GFP were directly fixed, DAPI stained and analysed by confocal microscopy. DAPI staining was used to define heterochromatin regions (white outlines). Scale bars, 2 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: After KBrO3 OGG1 is relocalized to foci resistant to detergent-containing buffer. (A) Subcellular fractionation of NT and KBrO3-treated OGG1–GFP cells. Cells were separated into fractions S1 (soluble proteins) and P1 (chromatin fraction) and different fractions analysed by western blot using an anti-GFP antibody. Lamin B1 was used as a loading control. (B) Distribution patterns of OGG1–GFP in NT and KBrO3-treated cells. Prior to fixation, soluble proteins were removed with CSK-0.5% triton when indicated. (C) NT cells expressing OGG1–GFP were directly fixed, DAPI stained and analysed by confocal microscopy. DAPI staining was used to define heterochromatin regions (white outlines). Scale bars, 2 µm.
Mentions: To determine OGG1 localization during the repair period, after the 30 min KBrO3 treatment cells were allowed to recover for 3 h in fresh medium before proceeding to subcellular fractionation. The soluble pool of proteins (S1), corresponding to the cytoplasm and nucleoplasm, was obtained by washing the cells with CSK buffer containing 0.5% triton. The resulting pellet (P1) corresponded to chromatin- and matrix-associated proteins. As revealed by western blot analysis of the S1 fraction, the majority of OGG1 remained soluble after KBrO3. However, whereas no retention of OGG1 could be observed in NT cells, KBrO3 induced the association of a fraction of hOGG1 with chromatin and nuclear matrix (Figure 2A).Figure 2.

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