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

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.

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.

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.

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.

Figure 4.

APE1 and XRCC1, together with OGG1 are relocalized to euchromatin in KBrO₃-treated cells. Fusion proteins (OGG1–DsRed and APE1–GFP or XRCC1–YFP) were transiently expressed in HeLa cells. Soluble proteins were extracted by CSK washes prior to fixation and DAPI staining. (A) NT cells, Scale bar=10 µm. (B) Three hours after KBrO₃ treatment. Plot profiles along the lines in the merged image reflect the co-localization of OGG1 with APE1 and XRCC1, respectively. Scale bar 2 µm. Fluorescence intensities of each channel along a line in the merged image are represented in the right panels.

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.

Figure 6.

Chromatin condensation by hypertonic shock impedes 8-oxoG repair. (A) NT cells pre-incubated or not with 250 mM sucrose for 3 h and washed with CSK–0,5% triton buffer prior to fixation. DNA is stained with DAPI. (B) OGG1–GFP cells are treated with 40 mM KBrO₃ for 30 min and allowed to recover in DMEM supplemented with 250 mM sucrose. In the last row of images, sucrose was removed after 3 h and replaced by fresh medium for 3 h. Cells were washed with CSK buffer prior to fixation. Graph represents GFP intensity for each condition (number of nucleus > 10). Scale bar = 10 µm. (C) 8-OxoG quantification by alkaline elution of cells treated with 40 mM KBrO₃ and recovered in 250 mM sucrose supplemented medium. Bilateral Student test (*P > 0.1 compared to the point 0; **P< 0.04 and P< 0.02 compared to the points 0 and 3 h sucrose respectively). (D) western blot of cells treated with 40 mM KBrO₃ and recovered in 250 mM sucrose supplemented medium. Antibodies against GFP are used and lamin B1 is used as a loading control. Quantification of signals using Scan GBox is represented on the graph on the right. Bilateral Student’s test (*P < 0.05).

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.