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To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX 77555. Tel.: 409-772-9414; Fax: 409-747-6869
Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555Departments of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555
* This work was supported, in whole or in part, by National Institutes of Health Grants RO1 ES018948 (to I. B.) from the NIEHS, 021830 (to I. B.) from the NIA/AG, and NIAID/AI062885-01 (to I. B.) from the NIAID and the Grants N01HV00245 (to Dr. A. Kurosky) and R01CA81063 (to S. M.) from the NHLBI Proteomic Center. This article contains supplemental Figs. S1–S7. 2 An Environmental Toxicology Research Training Fellow (Grant T32 ES007254 from the NIEHS). 3 Present address: Institute of Sport Science, Semmelweis University, H-1085 Budapest, Hungary. 4 Present address: Institute of Immunology, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, Hungary.
8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.
Reactive oxygen species, generated both endogenously during respiration and by various oxidases and environmental insults, induce multiple types of damage in the genome, including strand breaks and several types of oxidized bases (
), suggesting that alternative, compensatory enzymes could also repair 8-oxoG, at least in the active genes. On the other hand, Ogg1-deficient mice are resistant to inflammation, implicating OGG1 in proinflammatory signaling (
). We thus hypothesized a DNA repair-independent function of OGG1 that depends on the free 8-oxoG base. We report here that the OGG1·8-oxoG complex activates canonical Ras family GTPases, cellular signaling, and gene expression. We thus document for the first time a distinct, cell-signaling function of a DNA repair enzyme.
RESULTS AND DISCUSSION
The free 8-oxoG base is generated exclusively during the repair of 8-oxoG in DNA, initiated by OGG1 (
). To mimic a transient increase in its intracellular level, we added 8-oxoG base to OGG1-proficient cells (MRC5) and analyzed the impact on the transcriptome using Affymetrix GeneChip. Ingenuity Pathways Analysis of microarray gene expression data (National Center for Biotechnology Information (NCBI), GEO accession number GSE26813) showed that 8 of the top 10 pathways that responded to 8-oxoG involved the small G protein Ras (supplemental Fig. S1). Ras GTPases activate a variety of cellular signaling pathways (
To confirm these observations, we showed that the addition of 8-oxoG increased GTP-bound Ras levels in a time- (Fig. 1A) and dose-dependent (Fig. 1B) manner. The lowest dose of 8-oxoG base that increased Ras-GTP to a detectable level was 100 nm in MRC5 cells (Fig. 1B). The time course of Ras activation was consistent with rapid cellular uptake of 8-oxoG base. For example, at 1 min after the addition, ∼70% of 8-oxoG was taken up by the cells, as shown by GS/MS analysis (supplemental Fig. S2A). Importantly, after intranasal challenge of mice (
) with 8-oxoG (60 μl of 1 μm 8-oxoG), we observed increased Ras-GTP levels in the lungs (Fig. 1E).
We hypothesized that 8-oxoG bound to OGG1 mediates guanine nucleotide exchange in Ras. In support of this idea, adding 8-oxoG to OGG1-depleted MRC5 (Fig. 1C; supplemental Fig. S2, B and D) or HeLa-S cells (Fig. 1D; supplemental Fig. S2, C and D) did not cause an increase in Ras-GTP levels. Free 8-oxoG was unique in increasing the Ras-GTP level as neither 8-oxodG nor other oxidized bases (FapyG or 8-oxoA) nor the original guanine base displayed this activity (Fig. 1F).
The binding of free 8-oxoG base to OGG1 was analyzed by changes in the intrinsic Trp fluorescence of OGG1 (
). A concentration-dependent decrease in Trp fluorescence (Fig. 2A) indicated the OGG1 conformational change as a result of the interaction. The binding constant (Kd) 0.56 ± 0.19 nm calculated from the binding isotherms (Fig. 2B) indicates its high affinity for 8-oxoG, which was unexpected, and predicted that the product binding would inhibit the enzyme. However, we observed the exact opposite in that 8-oxoG stimulated the activity of OGG1 in a concentration-dependent manner (supplemental Fig. S3A). This suggests that 8-oxoG serves as a cofactor for OGG1 by binding to an independent site and not to the active site pocket in OGG1. This was further supported by our observation that the free FapyG base, with abundance similar to that of 8-oxoG in oxidatively damaged DNA, and which is an equally good OGG1 substrate (
) and observed that in the presence of 8-oxoG, OGG1 specifically was bound to H-Ras (Fig. 2C, lane 2). However, OGG1 alone did not interact with H-Ras under identical conditions (Fig. 2C, lane 1), suggesting that an 8-oxoG-induced conformational change in OGG1 (Fig. 2A) allows its binding to Ras. Quantitation of eluted OGG1 and comparison with input Ras indicated a nearly equimolar binding of OGG1 to H-Ras (Fig. 2D). Furthermore, GTP was more effective than GDP in inhibiting the interaction between Ras and OGG1·8-oxoG (Fig. 2E). These data strongly suggest that the conformation of nucleotide-free Ras allows the most stable interaction with OGG1·8-oxoG, which is weakened in the presence of guanine nucleotides. Similar interactions of K-Ras and N-Ras with OGG1·8-oxoG were also observed, and guanine nucleotides, especially GTP, decreased these interactions (supplemental Fig. S4, A and B). Our observations are consistent with those showing high affinity binding between nucleotide-free Ras and GEF (e.g. CDC25), which is decreased to an undetectable level by guanine nucleotides, especially GTP, due to nucleotide-induced conformational changes in the Ras protein (
Increases in the Ras-GTP level upon exposure of cells to 8-oxoG (Fig. 1) and physical interaction between Ras and OGG1 (Fig. 2, C, D, and E) could cause guanine nucleotide exchange. Indeed, in the presence of 8-oxoG, OGG1 caused replacement of GDP-bound to Ras with GTP (Fig. 2F) at equimolar or higher molar ratios of H-Ras:OGG1. We subsequently showed that OGG1 also catalyzed the release of H-Ras-bound GTP replacement with GDP (Fig. 2G). OGG1 or 8-oxoG alone did not induce guanine nucleotide exchange (Fig. 2, F and G). Densitometric analysis of the bands in Fig. 2, F and G (left panels) shows striking similarities between the kinetics of GDP-GTP and GTP-GDP exchange on Ras (Fig. 2, F and G, right panels), suggesting that OGG1 indiscriminately releases the nucleotide in vitro and allows rebinding; thus its activity is similar to that of other Ras-GEFs (
), and its subsequent phosphorylation is necessary, but not sufficient, for mediating the mitogen-activated protein kinase (MAPK) activity of Raf1 as phosphorylated Raf1 requires additional protein-protein and membrane-lipid interactions (
). Increasing the cellular 8-oxoG level in MRC5 cells induced rapid phosphorylation of the MAPK kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2) and the nuclear translocation of the latter (Fig. 3, A and B). To verify that ERK1/2 phosphorylation is Ras-dependent, H-, K-, and N-Ras were depleted with siRNA (Fig. 3C, upper panel). After 8-oxoG addition, ERK1/2 phosphorylation was significantly decreased in N-Ras-ablated MRC5 cells (Fig. 3C, middle panel, last lane).
Immunoblotting analysis showed abundant expression of N-Ras and K-Ras in MRC5 cells, whereas H-Ras was barely detectable (Fig. 3D, middle panel). Importantly, the addition of 8-oxoG to these cells resulted almost exclusively in N-Ras activation (Fig. 3D, upper panels), demonstrating selectivity to the activation process. These data are consistent with those showing that siRNA to N-Ras decreased ERK1/2 phosphorylation, whereas only a marginal effect of K-Ras depletion was seen (Fig. 3C, middle panel). To test whether 8-oxoG activates only N-Ras or other isoforms as well, we examined HeLa-S cells, which express H-Ras, K-Ras, and N-Ras (supplemental Fig. 6A). After 8-oxoG addition, H-, K-, and N-Ras were all activated (supplemental Fig. 6B), which implies that the 8-oxoG base activates Ras isoforms in a cell type-specific manner in OGG1-proficient cells.
In conclusion, we document for the first time that OGG1 binds the free 8-oxoG base at a nonsubstrate site with high affinity and that this complex interacts with the canonical Ras family GTPases to increase their GTP-bound forms by facilitating guanine nucleotide exchange. OGG1-mediated Ras activation initiates signal transduction for transcriptional activation of downstream genes. We propose that 8-oxoG released from DNA by OGG1 binds back to the enzyme to activate signaling pathways including those that may modulate expression of enzymes that inhibit 8-oxoG-induced mutagenesis by activating or preventing 8-oxoG incorporation into DNA from the nucleotide pool. We thus tested whether OGG1·8-oxoG-induced signaling affects the expression of MutT homolog-1 (MTH1) and MTH2 isoforms, the human homologs of Escherichia coli MutT, which hydrolyzes 8-oxodGTP (
). MTH1 but not the MTH2 gene was activated in MRC5 cells at 6 h after treatment with 8-oxoG (supplemental Fig. S7). Whether 8-oxoG similarly activates DNA repair in vivo has not yet been investigated. In any case, our results so far suggest an unusual, complex signaling network activated by small GTPases and triggered by the generation and repair of 8-oxoG in the genome.
We thank Dr. David Konkel (Biochemistry and Molecular Biology) for careful editing of the manuscript. We thank Drs. Miral Dizdaroglu and Pawel Jaruga (Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD) for assessment of 8-oxoG base levels. We also thank the anonymous reviewer for suggesting examination of the effect of 8-oxoG addition on MTH expression.