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, 286 (52), 44679-90

Poly(ADP-ribose) Polymerase 1 (PARP-1) Binds to 8-oxoguanine-DNA Glycosylase (OGG1)

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Poly(ADP-ribose) Polymerase 1 (PARP-1) Binds to 8-oxoguanine-DNA Glycosylase (OGG1)

Nicole Noren Hooten et al. J Biol Chem.

Abstract

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.

Figures

FIGURE 1.
FIGURE 1.
Identification of proteins binding to OGG1. A, Coomassie colloidal blue staining of purified GST and GST-OGG1 (20 μg) proteins is shown. B, nuclear extracts from mouse brain and liver were immunoblotted with the indicated antibodies (cyt) cytosolic fraction (nuc) nuclear fraction. C, GST-OGG1 and GST control were used to precipitate proteins from mouse brain and liver nuclear extracts (NE). GST precipitations were analyzed on a 12% polyacrylamide gel and silver-stained. −, GST fusion protein alone. pdown, pulldown. D, GST-OGG1 was used to precipitate proteins from mouse brain and liver. Samples were analyzed on an 8% polyacrylamide gel and stained with Coomassie colloidal blue. The arrow points to the band, corresponding to PARP-1, that was excised and analyzed by mass spectrometry. −, GST fusion protein alone.
FIGURE 2.
FIGURE 2.
OGG1 binds directly to PARP-1. A, mouse liver nuclear extracts were incubated with immobilized GST or GST-OGG1 and were mock-treated (−) or treated (+) with DNase1. The proteins remaining associated with the GST fusion protein were probed with anti-PARP-1 antibodies. The amount of GST-OGG1 in the precipitations was determined using anti-OGG1 antibodies. NE, nuclear extracts. B and C, binding to GST-OGG1 or GST was assessed using an in vitro binding assay. GST-OGG1 or GST control (1 μg) were incubated with purified PARP-1 (0.25 μg), and samples were immunoblotted with anti-PARP-1 antibodies and reprobed with anti-OGG1 antibodies (C) or stained with Ponceau S (B) as loading controls. The arrow indicates GST-OGG1. C, GST-OGG1 incubated with PARP-1 in vitro was either mock-treated (−) or treated with DNase I (+). OGG1 retains binding to PARP-1 despite DNase treatment. D, HeLa cells transfected with FLAG vector control or FLAG-OGG1 were untreated (−) or treated for 30 min with 5 mm H2O2 (+). PARP-1 immunoprecipitates or lysates were probed with anti-FLAG antibodies or anti-PARP-1 antibodies. Increased binding of OGG1 to PARP-1 was also observed after treatment with lower concentrations of H2O2 (data not shown). E, schematic of GST-OGG1 fusion proteins is shown. HhH, Helix-hairpin-Helix; NLS, nuclear localization sequence; MLS, mitochondrial localization sequence. F, an in vitro binding assay was used to assess the binding of GST control, WT OGG1, and various fragments of OGG1 (1 μg) to purified PARP-1 (0.25 μg). GST precipitations were immunoblotted with anti-PARP-1 antibodies to identify PARP-1 binding and reprobed with anti-GST antibodies to visualize fusion proteins. G, a schematic of PARP-1 proteins is shown. H, purified His-tagged PARP-1 domains (1 μg) were incubated with GST-OGG1 (1 μg), and the precipitations were immunoblotted with anti-His antibodies and reprobed with anti-OGG1 antibodies to determine the amount of GST-OGG1 in the precipitations. The arrows indicate the bands corresponding to the DNA binding domain and the BRCT domain. Incubation with ethidium bromide (EthBr; +) abrogates the interaction between GST-OGG1 and the DNA binding domain.
FIGURE 3.
FIGURE 3.
OGG1 stimulates the poly(ADP-ribosyl)ation activity of PARP-1. A and B, PARP-1 activity was measured by determining the amount of PAR deposited on immobilized histones in an ELISA assay. The addition of OGG1 (0.5 μg) increased the amount of PAR synthesis by PARP-1 (2 ng). B, ELISA assays were performed with or without activated DNA. The histograms show the averages + S.E. from triplicate (A) and quadruplicate (B) experiments. **, p < 0.01 compared with untreated control by Student's t test for A. *, p < 0.05 and ***, p < 0.001 for the indicated comparisons in B using one-way analysis of variance and Tukey's post-hoc test.
FIGURE 4.
FIGURE 4.
Reduced levels of OGG1 impair poly(ADP-ribosyl)ation of cellular proteins after oxidative stress. A, WT or OGG1−/− MEFs were either control-treated or treated with 500 μm H2O2 for 10 min and stained with anti-PAR antibodies and DAPI. B, percentage of PAR-positive nuclei were calculated by counting the number of PAR-positive nuclei per DAPI-stained nuclei in the indicated cell lines. A total of ∼1000–1300 cells were counted from triplicate coverslips for each experiment. The histogram shows the averages normalized to WT MEFs ± S.E. from three independent experiments. **, p < 0.01 compared with control using Student's t test. C, WT or OGG1−/− MEFs were untreated (−) or treated for 10 min with 500 μm H2O2 (+). Lysates were probed with anti-PARP-1 antibodies and anti-GAPDH antibodies as a protein loading control. The relative levels of PARP-1 were not significantly different between WT (1.0) and OGG1−/− MEFs (1.07). The numbers in parentheses show the average relative levels of PARP-1 normalized to GAPDH and to WT cells from three independent experiments. D, shown is accumulation of DNA damage in OGG1−/− cells in response to H2O2 treatment. WT or OGG1−/− MEFs were untreated or treated with 100 μm H2O2 for 30 min. Oxidative DNA damage was analyzed using the alkaline comet assay. The histogram represents the mean of five independent experiments ± S.E. *, p < 0.05 comparing OGG1−/− to WT using Student's t test.
FIGURE 5.
FIGURE 5.
PARP-1 inhibits OGG1 activity. A, recombinant OGG1 (320 nm) was incubated with buffer, PARP-1 cocktail (PARP-1 ct), PARP-1 (180 nm), PAR, or PARP-1 and PARP-1 cocktail and reacted with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG/C mismatch. After a 30-min incubation at 37 °C, the cleavage products were analyzed using 20% denaturing gels and phosphorimaging. A representative experiment is shown in the left panel, and the histogram represents the mean ± S.E. from four independent experiments. The percent incision was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of uncleaved substrate (top band). The data were normalized to the incision activity of OGG1 alone (100%). B, incision assays were performed as in A with the exception that different concentrations of PARP-1 with or without PARP cocktail were added to the OGG1 (320 nm) incision assay. The percent incision was calculated as above, and the histogram represents the mean ± S.E. from five independent experiments for PARP-1 alone and three independent experiments for activated PARP-1. C, different concentrations of OGG1 were incubated with or without PARP-1 (180 nm), and the incision assays were performed as in A. Incision was calculated by taking the amount of cleaved substrate (lower band of A) normalized to the amount of uncleaved substrate (top band of A). The histogram represents the mean ± S.E. from three independent experiments. For A–C, *, p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparisons using one-way analysis of variance and Tukey's post-hoc test. D, HeLa cells were untreated or treated with menadione (25 μm), ABT-888 (5 μm), or both menadione and ABT-888. Cells were stained with anti-8-oxoG antibodies and DAPI as described under “Experimental Procedures.”
FIGURE 6.
FIGURE 6.
Binding of OGG1 polymorphic variants to PARP-1. A, shown is Coomassie colloidal blue staining of GST, WT OGG1, or OGG1 with the indicated amino acid mutations. Precipitations with the indicated GST fusion proteins were performed from liver nuclear extracts (NE; B) or with purified PARP-1 alone (C). Samples were probed with anti-PARP-1 antibodies. Anti-OGG1 antibodies were used to visualize the amount of GST-OGG1 fusion proteins. In C, the lane indicated WT alone indicates the WT fusion protein without the addition of PARP-1. D, an ELISA assay was used to measure the amount of PAR deposited by PARP-1 on immobilized histones. WT, R229Q, or S326C (0.5 μg) recombinant proteins were incubated with PARP-1 (2 ng) for 6 min before the addition of activated DNA and PARP-1 cocktail. The histograms show the averages ± S.E. from triplicate experiments. *, p < 0.05 and **, p < 0.01 for the indicated comparisons using one-way analysis of variance and Tukey's post-hoc test.
FIGURE 7.
FIGURE 7.
Loss of OGG1 and inhibition of PARP-1 impairs colony formation and cell survival in response to DNA damage. A, colony formation assay of MEFs either untreated (Con) or treated with H2O2, ABT-888 or both H2O2 and ABT-888 are shown. B, cell survival of MEFs was measured after incubation for 24 h with the indicated treatments. Both histograms show the normalized averages ± S.E. from five independent experiments. *, p < 0.05 and ***, p < 0.001 comparing OGG1−/− to WT using one-way analysis of variance and Tukey's post-hoc test.

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