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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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OGG1 is excluded from heterochromatin and colocalizes with euchromatin-associated proteins. Following a 3-h recovery after KBrO3 treatment, soluble proteins were removed with CSK buffer prior to fixation and analysis by confocal microscopy. (A) DNA (upper panel) or RNA (lower panel) were digested before fixation and PI staining. Solid arrows indicate ribosomal RNA in nucleoli. Open arrows show patches of heterochromatin. Positions of the line scans used for the plot profile are indicated in the merged images. (B) Heterochromatin was immunostained with HP1α (upper panel) and H3meK9 (lower panel), in red. Cytofluorogram of both merged images shows a great dispersion of points, reflecting an absence of correlation of both intensity signals. (C) RNA polymerase II (upper panel) and H3meK4 (lower panel) partially colocalize with OGG1–GFP (filled arrows), although some OGG1 foci are excluded from RNA polymerase II staining (unfilled arrows). Correlations between green and red signals are presented in the cytofluorograms. (D) In situ hybridization of mRNA with oligo(dT)5’Cy3 in NT and KBrO3-treated cells. Line scans used for the plot profiles are indicated in the merged images. Both plot profiles and cytofluorogram show a colocalization between polyadenylated RNA and OGG1 after KBrO3 and an exclusion of both signals in NT cells. (E) Heterochromatin/euchromatin fractionation of KBrO3-treated cells. Heterochromatin (HP1α) and euchromatin (H3meK4 and RNA polymerase II) markers are used as controls. Scale bars, 2 µm.
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Figure 5: OGG1 is excluded from heterochromatin and colocalizes with euchromatin-associated proteins. Following a 3-h recovery after KBrO3 treatment, soluble proteins were removed with CSK buffer prior to fixation and analysis by confocal microscopy. (A) DNA (upper panel) or RNA (lower panel) were digested before fixation and PI staining. Solid arrows indicate ribosomal RNA in nucleoli. Open arrows show patches of heterochromatin. Positions of the line scans used for the plot profile are indicated in the merged images. (B) Heterochromatin was immunostained with HP1α (upper panel) and H3meK9 (lower panel), in red. Cytofluorogram of both merged images shows a great dispersion of points, reflecting an absence of correlation of both intensity signals. (C) RNA polymerase II (upper panel) and H3meK4 (lower panel) partially colocalize with OGG1–GFP (filled arrows), although some OGG1 foci are excluded from RNA polymerase II staining (unfilled arrows). Correlations between green and red signals are presented in the cytofluorograms. (D) In situ hybridization of mRNA with oligo(dT)5’Cy3 in NT and KBrO3-treated cells. Line scans used for the plot profiles are indicated in the merged images. Both plot profiles and cytofluorogram show a colocalization between polyadenylated RNA and OGG1 after KBrO3 and an exclusion of both signals in NT cells. (E) Heterochromatin/euchromatin fractionation of KBrO3-treated cells. Heterochromatin (HP1α) and euchromatin (H3meK4 and RNA polymerase II) markers are used as controls. Scale bars, 2 µm.

Mentions: We next explored the possibility that OGG1 would be recruited to specific chromatin domains. When KBrO3-treated cells were submitted to DNAse digestion prior to fixation, the OGG1–GFP signal was still observed, suggesting that while processing DNA OGG1 is associated with an insoluble nuclear fraction. Similar observations have been reported for proteins associated with transcription and replication factories. Surprisingly, OGG1 perfectly co-localized with the PI signal, suggesting an association with RNA-rich regions of the nucleus (Figure 5A, upper panel). However, RNA digestion prior to CSK buffer wash and fixation also failed to remove the OGG1–GFP signal in KBrO3-treated cells (Figure 5A, lower panel). Interestingly, the OGG1 signal was excluded from the large patches of heterochromatin revealed by PI staining after RNAse digestion. Indeed, line scans of OGG1 (green) and PI (red) staining on Figure 5A clearly showed a complete exclusion of the DNA glycosylase from DNA-dense regions (lower panel). Taken together, these results show that KBrO3 treatment induces an active relocalization of OGG1 from a soluble pool to less-condensed DNA regions enriched in RNA.Figure 5.


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)

OGG1 is excluded from heterochromatin and colocalizes with euchromatin-associated proteins. Following a 3-h recovery after KBrO3 treatment, soluble proteins were removed with CSK buffer prior to fixation and analysis by confocal microscopy. (A) DNA (upper panel) or RNA (lower panel) were digested before fixation and PI staining. Solid arrows indicate ribosomal RNA in nucleoli. Open arrows show patches of heterochromatin. Positions of the line scans used for the plot profile are indicated in the merged images. (B) Heterochromatin was immunostained with HP1α (upper panel) and H3meK9 (lower panel), in red. Cytofluorogram of both merged images shows a great dispersion of points, reflecting an absence of correlation of both intensity signals. (C) RNA polymerase II (upper panel) and H3meK4 (lower panel) partially colocalize with OGG1–GFP (filled arrows), although some OGG1 foci are excluded from RNA polymerase II staining (unfilled arrows). Correlations between green and red signals are presented in the cytofluorograms. (D) In situ hybridization of mRNA with oligo(dT)5’Cy3 in NT and KBrO3-treated cells. Line scans used for the plot profiles are indicated in the merged images. Both plot profiles and cytofluorogram show a colocalization between polyadenylated RNA and OGG1 after KBrO3 and an exclusion of both signals in NT cells. (E) Heterochromatin/euchromatin fractionation of KBrO3-treated cells. Heterochromatin (HP1α) and euchromatin (H3meK4 and RNA polymerase II) markers are used as controls. Scale bars, 2 µm.
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Figure 5: OGG1 is excluded from heterochromatin and colocalizes with euchromatin-associated proteins. Following a 3-h recovery after KBrO3 treatment, soluble proteins were removed with CSK buffer prior to fixation and analysis by confocal microscopy. (A) DNA (upper panel) or RNA (lower panel) were digested before fixation and PI staining. Solid arrows indicate ribosomal RNA in nucleoli. Open arrows show patches of heterochromatin. Positions of the line scans used for the plot profile are indicated in the merged images. (B) Heterochromatin was immunostained with HP1α (upper panel) and H3meK9 (lower panel), in red. Cytofluorogram of both merged images shows a great dispersion of points, reflecting an absence of correlation of both intensity signals. (C) RNA polymerase II (upper panel) and H3meK4 (lower panel) partially colocalize with OGG1–GFP (filled arrows), although some OGG1 foci are excluded from RNA polymerase II staining (unfilled arrows). Correlations between green and red signals are presented in the cytofluorograms. (D) In situ hybridization of mRNA with oligo(dT)5’Cy3 in NT and KBrO3-treated cells. Line scans used for the plot profiles are indicated in the merged images. Both plot profiles and cytofluorogram show a colocalization between polyadenylated RNA and OGG1 after KBrO3 and an exclusion of both signals in NT cells. (E) Heterochromatin/euchromatin fractionation of KBrO3-treated cells. Heterochromatin (HP1α) and euchromatin (H3meK4 and RNA polymerase II) markers are used as controls. Scale bars, 2 µm.
Mentions: We next explored the possibility that OGG1 would be recruited to specific chromatin domains. When KBrO3-treated cells were submitted to DNAse digestion prior to fixation, the OGG1–GFP signal was still observed, suggesting that while processing DNA OGG1 is associated with an insoluble nuclear fraction. Similar observations have been reported for proteins associated with transcription and replication factories. Surprisingly, OGG1 perfectly co-localized with the PI signal, suggesting an association with RNA-rich regions of the nucleus (Figure 5A, upper panel). However, RNA digestion prior to CSK buffer wash and fixation also failed to remove the OGG1–GFP signal in KBrO3-treated cells (Figure 5A, lower panel). Interestingly, the OGG1 signal was excluded from the large patches of heterochromatin revealed by PI staining after RNAse digestion. Indeed, line scans of OGG1 (green) and PI (red) staining on Figure 5A clearly showed a complete exclusion of the DNA glycosylase from DNA-dense regions (lower panel). Taken together, these results show that KBrO3 treatment induces an active relocalization of OGG1 from a soluble pool to less-condensed DNA regions enriched in RNA.Figure 5.

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