Limits...
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.

Show MeSH

Related in: MedlinePlus

OGG1 is dynamically recruited to chromatin after KBrO3 treatment. (A) Confocal analysis of OGG1–GFP recruitment to chromatin fraction in KBrO3-treated cells. Cells were allowed to recover in fresh medium for the indicated times before CSK washing for the removal of the soluble fraction. Scale bars, 10 µm. (B) Fluorescence intensities of cells showed in (A). Values represent an average of fluorescence intensity of cells (n > 50). (C) Upper panel, western blot with an anti-GFP antibody shows OGG1–GFP transient accumulation in the chromatin fraction (P1) after KBrO3 treatment. Sm was used as a loading control. The lower panel shows the accumulation of overexpressed and endogenous OGG1 proteins in the nuclear matrix fraction obtained after DNAse digestion. OGG1–GFP and endogenous OGG1 were visualized with anti-GFP and anti-OGG1 (PA3) primary antibodies, respectively. Lamin B1 served as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875005&req=5

Figure 3: OGG1 is dynamically recruited to chromatin after KBrO3 treatment. (A) Confocal analysis of OGG1–GFP recruitment to chromatin fraction in KBrO3-treated cells. Cells were allowed to recover in fresh medium for the indicated times before CSK washing for the removal of the soluble fraction. Scale bars, 10 µm. (B) Fluorescence intensities of cells showed in (A). Values represent an average of fluorescence intensity of cells (n > 50). (C) Upper panel, western blot with an anti-GFP antibody shows OGG1–GFP transient accumulation in the chromatin fraction (P1) after KBrO3 treatment. Sm was used as a loading control. The lower panel shows the accumulation of overexpressed and endogenous OGG1 proteins in the nuclear matrix fraction obtained after DNAse digestion. OGG1–GFP and endogenous OGG1 were visualized with anti-GFP and anti-OGG1 (PA3) primary antibodies, respectively. Lamin B1 served as a loading control.

Mentions: In order to determine the kinetics of OGG1 recruitment to chromatin after KBrO3 treatment, cells expressing OGG1–GFP were washed with CSK after different recovery times prior to analysis by confocal microscopy. As soon as 30 min after treatment, 70–90% of the cells showed a detergent-resistant OGG1 fraction that increased with time to peak between 3 and 4 h after the end of the treatment (Figure 3A and B). The fraction of OGG1 associated with chromatin subsequently decreased to reach basal levels after 8 h of recovery. Subcellular fractionation and immunoblotting analysis confirmed the recruitment kinetics (Figure 3C, upper panel) and allowed to establish that the endogenous OGG1 was also recruited to the insoluble fraction with similar kinetics (Figure 3C, lower panel). Interestingly, the maximum accumulation of OGG1 in the chromatin fraction coincided with the maximum rate of repair of 8-oxoG (Figure 1C), suggesting a link between BER initiated by the DNA glycosylase and its recruitment to chromatin.Figure 3.


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 dynamically recruited to chromatin after KBrO3 treatment. (A) Confocal analysis of OGG1–GFP recruitment to chromatin fraction in KBrO3-treated cells. Cells were allowed to recover in fresh medium for the indicated times before CSK washing for the removal of the soluble fraction. Scale bars, 10 µm. (B) Fluorescence intensities of cells showed in (A). Values represent an average of fluorescence intensity of cells (n > 50). (C) Upper panel, western blot with an anti-GFP antibody shows OGG1–GFP transient accumulation in the chromatin fraction (P1) after KBrO3 treatment. Sm was used as a loading control. The lower panel shows the accumulation of overexpressed and endogenous OGG1 proteins in the nuclear matrix fraction obtained after DNAse digestion. OGG1–GFP and endogenous OGG1 were visualized with anti-GFP and anti-OGG1 (PA3) primary antibodies, respectively. Lamin B1 served as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875005&req=5

Figure 3: OGG1 is dynamically recruited to chromatin after KBrO3 treatment. (A) Confocal analysis of OGG1–GFP recruitment to chromatin fraction in KBrO3-treated cells. Cells were allowed to recover in fresh medium for the indicated times before CSK washing for the removal of the soluble fraction. Scale bars, 10 µm. (B) Fluorescence intensities of cells showed in (A). Values represent an average of fluorescence intensity of cells (n > 50). (C) Upper panel, western blot with an anti-GFP antibody shows OGG1–GFP transient accumulation in the chromatin fraction (P1) after KBrO3 treatment. Sm was used as a loading control. The lower panel shows the accumulation of overexpressed and endogenous OGG1 proteins in the nuclear matrix fraction obtained after DNAse digestion. OGG1–GFP and endogenous OGG1 were visualized with anti-GFP and anti-OGG1 (PA3) primary antibodies, respectively. Lamin B1 served as a loading control.
Mentions: In order to determine the kinetics of OGG1 recruitment to chromatin after KBrO3 treatment, cells expressing OGG1–GFP were washed with CSK after different recovery times prior to analysis by confocal microscopy. As soon as 30 min after treatment, 70–90% of the cells showed a detergent-resistant OGG1 fraction that increased with time to peak between 3 and 4 h after the end of the treatment (Figure 3A and B). The fraction of OGG1 associated with chromatin subsequently decreased to reach basal levels after 8 h of recovery. Subcellular fractionation and immunoblotting analysis confirmed the recruitment kinetics (Figure 3C, upper panel) and allowed to establish that the endogenous OGG1 was also recruited to the insoluble fraction with similar kinetics (Figure 3C, lower panel). Interestingly, the maximum accumulation of OGG1 in the chromatin fraction coincided with the maximum rate of repair of 8-oxoG (Figure 1C), suggesting a link between BER initiated by the DNA glycosylase and its recruitment to chromatin.Figure 3.

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