New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5'-cyclopurine 2'-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC-HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients.     

8-Hydroxyguanine levels and repair capacity during mouse embryonic stem cell differentiation

To evaluate the defence capacities of embryonic stem (ES) cells against gene impairment, this study measured the levels of 8-hydroxyguanine (8-OH-Gua), a well-known marker of oxidative stress in DNA, and its repair capacity during differentiation. Undifferentiated ES cells (EB3) were cultured without leukaemia inhibitory factor (LIF) for 0, 4 and 7 days and are referred to as ES-D0, ES-D4 and ES-D7, respectively. These three cell lines were treated with 300 μM hydrogen peroxide (H(2)O(2)) for 48 and 72 h. After treatment, the amounts of 8-OH-Gua in the cells were determined by the high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) method. The levels of 8-OH-Gua in ES-D7 treated with H(2)O(2) were higher than those in ES-D0 and ES-D4, suggesting that the DNA in the undifferentiated cells was protected against gene impairment, as compared to that in the differentiated cells. To examine the repair capacity for 8-OH-Gua, this study analysed the expression of 8-OH-Gua repair-associated genes, 8-oxoguanine DNA glycosylase 1 (OGG1), MutY homolog (MUTYH) and Mut T homolog 1 (MTH1), in ES-D0, ES-D4 and ES-D7. The mRNA levels of MUTYH and MTH1 showed no significant change, whereas OGG1 mRNA was significantly decreased in ES-D7 treated with H(2)O(2). Moreover, it was observed that ES-D7 treated with H(2)O(2) readily underwent apoptosis, in comparison to its undifferentiated counterparts, ES-D0 and ES-D4. Taken together, ES cells are more resistant to DNA oxidative stresses than differentiated cells.     

Excision repair of 8-hydroxyguanine in mammalian cells: the mouse Ogg1 protein as a model

8-Hydroxyguanine (8-OH-Gua) is a major mutagenic lesion produced on DNA by the oxidative stress induced by either the endogen metabolism or the exposure to external agents. In bacteria and yeast this modified base can be removed by specific DNA glycosylases. Recently a human gene coding for an 8-OH-Gua DNA glycosylase/AP lyase has been identified by its homology to the yeast OGG1. This gene is located in human chromosome 3p25, a region commonly rearranged in various cancers, specially in lung tumor cells. We report here the cloning, by sequence homology to the yeast OGG1, of a mouse cDNA coding for a 8-OH-Gua DNA glycosylase with 84% and 38% identity to the human and yeast relevant proteins, respectively. The Ogg1 gene is localized to the mouse chromosome 6E. The mouse Ogg1 cDNA, when expressed in Escherichia coli, is capable of suppressing the spontaneous mutator phenotype of a DNA repair deficient fpg mutY strain. The mouse Ogg1 protein acts efficiently on duplexes in which the 8-OH-Gua is paired with a cytosine but is inactive on 8-OH-Gua:Ade pair, consistently with its proposed biological role in the avoidance of mutations. A comparison of the mouse enzyme with other eukaryotic Ogg1 enzymes is also presented. The isolation of this gene will allow the development of an animal model to study the effects of oxidative stress on carcinogenesis and degenerative diseases.     

Interaction of Sirt3 with OGG1 contributes to repair of mitochondrial DNA and protects from apoptotic cell death under oxidative stress

Sirtuin 3 (Sirt3), a major mitochondrial NAD⁺-dependent deacetylase, targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response. In this study, we identified the human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged genome, as a new target protein for Sirt3. We found that Sirt3 physically associated with OGG1 and deacetylated this DNA glycosylase and that deacetylation by Sirt3 prevented the degradation of the OGG1 protein and controlled its incision activity. We further showed that regulation of the acetylation and turnover of OGG1 by Sirt3 played a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress. We observed that following ionizing radiation, human tumor cells with silencing of Sirt3 expression exhibited deteriorated oxidative damage of mtDNA, as measured by the accumulation of 8-oxoG and 4977 common deletion, and showed more severe mitochondrial dysfunction and underwent greater apoptosis in comparison with the cells without silencing of Sirt3 expression. The results reported here not only reveal a new function and mechanism for Sirt3 in defending the mitochondrial genome against oxidative damage and protecting from the genotoxic stress-induced apoptotic cell death but also provide evidence supporting a new mtDNA repair pathway.     

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.Oxidative DNA damage is generated at high levels in mammalian cells, even in cells not exposed to exogenous sources of reactive oxygen species. Several kinds of DNA modifications are formed upon oxidative stress (8). The most prevalent modifications, quantitatively, are single-strand breaks and oxidized bases. Clustered DNA damage, when two or more modifications are closely positioned in opposite strands, is detectable after gamma irradiation and has recently been shown to be generated by normal oxidative metabolism (3, 35). One unique aspect of such clustered lesions is that they can be converted into double-strand breaks (DSB) if a DNA glycosylase removes the two opposite bases and an apurinic/apyrimidinic (AP)-endonuclease cleaves the resulting abasic sites. Thus, although quantitatively minor, DSB are possible outcomes of oxidative DNA damage.Oxidized DNA bases are repaired primarily by the base excision repair pathway (BER) (22, 39). BER is initiated by a lesion-specific DNA N-glycosylase that recognizes and excises the damaged base. Eight-hydroxyguanine (8-oxoG) is one of the most abundant oxidized bases detected in cellular DNA. This adduct is easily bypassed by replicative polymerases; however, it can direct the misincorporation of adenine opposite 8-oxoG, thus leading to G·C-to-T·A transversion mutations (31). 8-oxoG accumulation has been causally associated with carcinogenesis and aging in several experimental models (1, 12). In eukaryotes, oxoguanine DNA glycosylase (OGG1) is the major 8-oxoG DNA glycosylase. OGG1 possesses an associated AP-lyase activity, such that it removes 8-oxoG and cleaves the DNA backbone. Human cells express two distinct OGG1 isoforms, α and β, which share the first 316 amino acids but differ significantly in their C termini (25). While OGG1-α is a bone fide DNA glycosylase (5) and localizes both to nuclei and mitochondria, OGG1-β localizes exclusively to mitochondria. We recently showed that the recombinant OGG1-β protein has no DNA glycosylase activity (13). The high degree of conservation of repair pathways for 8-oxoG, from bacteria to humans, along with epidemiological data correlating OGG1 polymorphisms and activity with predisposition to some cancers (11, 27, 33) attest to the biological importance of the repair of 8-oxoGs and other oxidative DNA lesions.Until recently, distinct classes of DNA lesions were believed to be metabolized by different and independent repair pathways. However, experimental evidence indicates that these pathways can interact and that there is a considerable degree of overlap in their substrate specificity and in the proteins that participate in each pathway. Experiments using yeast strains lacking one or more distinct DNA repair genes suggest that DSB repair pathways may play a role in repair of oxidative DNA damage. Swanson et al. showed that while yeast cells lacking ntg1 and ntg2 (homologues of Escherichia coli endonuclease III, a DNA glycosylase specific for pyrimidine lesions formed by oxidation) and apn1 (the major yeast abasic site endonuclease) are not overtly sensitive to oxidative stress, the additional disruption of the rad52 gene significantly increases sensitivity to H₂O₂ and menadione (36). Similarly, yeast cells expressing decreased levels of frataxin, which leads to elevated oxidative stress, show accumulation of oxidative damage in nuclear DNA only in a rad52 mutant background (18). RAD52 is a member of the RAD51 epistatic group. These proteins are believed to be involved in the early steps of homologous recombination, contributing to homology search and strand invasion; disruption of the corresponding genes renders cells deficient in DSB repair and hyper-recombinogenic (19).These results suggested a possible role for RAD52 in the repair of oxidative DNA damage. Moreover, an in vitro screening of protein partners that interact physically with OGG1-β performed in our lab (unpublished data) showed that human RAD52 strongly interacted with this glycosylase, again suggesting a possible function for RAD52 in the oxidative DNA damage response. Thus, we investigated whether RAD52 plays a role in the repair of oxidative DNA damage in human cells. We show here that human RAD52 physically interacts with both OGG1-α and -β, in vitro and in cell extracts. We also show that OGG1-α and -β inhibit RAD52 enzymatic activities. Conversely, RAD52 stimulates OGG1-α 8-oxoG incision activity. RAD52 colocalizes with OGG1-α in cells, and this colocalization increases after oxidative stress. Moreover, lower RAD52 expression, via gene knockdown (KD) or disruption of the RAD52 gene, render cells sensitive to oxidative stress. Based on our results, we discuss a model in which OGG1 and RAD52 cooperate to repair 8-oxoG lesions.     

Direct visualization of repair of oxidative damage by OGG1 in the nuclei of live cells

Oxidative DNA damage caused by intracellular reactive oxygen species (ROS) is widely considered to be important in the pathology of a range of human diseases including cancer as well as in the aging process. A frequently occurring mutagenic base lesion produced by ROS is 8-oxo deoxyguanine (8-oxo dG) and the major enzyme for repair of 8-oxo dG is 8-oxoguanine-DNA glycosylase 1 (OGG1). There is now substantial evidence from bulk biochemical studies that a common human polymorphic variant of OGG1 (Ser326Cys) is repair deficient, and this has been linked to individual risk of pathologies related to oxidative stress. In the current study, we have used the technique of multiphoton microscopy to induce highly localized oxidative DNA damage in discrete regions of the nucleus of live cells. Cells transfected with GFP-tagged OGG1 proteins demonstrated rapid (<2 min) accumulation of OGG1 at sites of laser-induced damage as indicated by accumulation of GFP-fluorescence. This was followed by repair as evidenced by loss of the localized fluorescence over time. Quantification of the rate of repair confirmed that the Cys326 variant of OGG1 is repair deficient and that the initial repair rate of damage by Cys326 OGG1 was 3 to 4 fold slower than that observed for Ser326 OGG1. These values are in good agreement with kinetic data comparing the Ser326 and Cys326 proteins obtained by biochemical studies.     

OGG1 is degraded by calpain following oxidative stress and cisplatin exposure

Deficient repair activity for 8-hydroxy-2'-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer's disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca(2+)-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (proline, glutamic acid, serine, threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion, and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

Acute arsenite-induced 8-hydroxyguanine is associated with inhibition of repair activity in cultured human cells

8-Hydroxyguanine (8-OH-Gua) is one of the major modified bases in DNA produced by oxidative damage. Human lung carcinoma cells (A549) were treated with 0.5-2mM sodium arsenite for 4h. By an immunohistochemical type procedure, 8-OH-Gua was clearly detected in A549 cells using a fluorescence microscope and an increase in the percentage of A549 cells with oxidative DNA damage was observed using flow cytometry. The formation of 8-OH-Gua in DNA was also detected by a HPLC-ECD. A dose-dependent increase in oxidative DNA damage in A549 cells with increasing arsenite concentrations was obtained. Therefore, oxidative stress is induced after arsenite treatment. Furthermore, we also found that arsenite decreased the activity of the 8-OH-Gua repair enzyme, hOGG1 (8-oxoguanine-DNA glycosylase 1) as well as its gene and protein expression. We conclude that the 8-OH-Gua level in cultured human cells increases partly by the generation of reactive oxygen species (ROS) and partly by the influence on hOGG1 expression, followed by the inhibition of the repair activity for 8-OH-Gua.     

OGG1 expression and OGG1 Ser326Cys polymorphism and risk of lung cancer in a prospective study

Oxidative DNA damage is believed to be implicated in lung carcinogenesis. 8-OxodG is a mutagenic and abundant oxidative modification induced in DNA. OGG1, NEIL1 and MUTYH are all involved in the repair and prevention of 8-oxodG-derived mutations and may be up-regulated by oxidative stress. The polymorphism OGG1 Ser326Cys has in some studies been associated with risk of lung cancer. In a population-based cohort of 57,053 Danes, we examined associations between mRNA levels of OGG1, NEIL1, MUTYH and NUDT in buffy coat material and subsequent lung cancer risk. 260 cases with lung cancer were identified and a sub-cohort of 263 individuals was matched on sex, age and smoking duration. We found that OGG1 mRNA levels in healthy individuals were not associated with risk of subsequent getting lung cancer. However, subjects with the OGG1 Cys326/Cys326 genotype had a higher expression level of OGG1 mRNA than wildtype-allele carriers. For homozygous Cys326 carriers, the incidence rate ratio (IRR) was 1.51 (95% CI: 1.09-2.08) for a doubling of the OGG1 mRNA level and there was a statistically significant interaction between the genotype and mRNA level. Among never-smokers, the IRR was 4.29 (1.09-16.9) per doubling of the OGG1 mRNA level, which was not found among smokers. Furthermore, we found a positive correlation between OGG1 mRNA expression and urinary excretion of 8-oxodG (RS=0.18; p<0.005). NUDT1 mRNA levels were omitted due to low and unreliable expression levels. The results suggest that OGG1 mRNA levels should be regarded as a biomarker of exposure to oxidative stress with induction of DNA rather than a marker of inborn DNA repair capacity.     

The defense mechanisms in mammalian cells against oxidative damage in nucleic acids and their involvement in the suppression of mutagenesis and cell death

To counteract oxidative damage in nucleic acids, mammalian cells are equipped with several defense mechanisms. We herein review that MTH1, MUTYH and OGG1 play important roles in mammalian cells avoiding an accumulation of oxidative DNA damage, both in the nuclear and mitochondrial genomes, thereby suppressing carcinogenesis and cell death. MTH1 efficiently hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP and 2-hydroxy (OH)-dATP, to the monophosphates, thus avoiding the incorporation of such oxidized nucleotides into the nuclear and mitochondrial genomes. OGG1 excises 8-oxoG in DNA as a DNA glycosylase and thus minimizes the accumulation of 8-oxoG in the cellular genomes. MUTYH excises adenine opposite 8-oxoG, and thus suppresses 8-oxoG-induced mutagenesis. MUTYH also possesses a 2-OH-A DNA glycosylase activity for excising 2-OH-A incorporated into the cellular genomes. Increased susceptibilities to spontaneous carcinogenesis of the liver, lung or intestine were observed in MTH1-, OGG1- and MUTYH-null mice, respectively. The increased occurrence of lung tumors in OGG1-null mice was abolished by the concomitant disruption of the Mth1 gene, indicating that an increased accumulation of 8-oxoG and/or 2-OH-A might cause cell death. Furthermore, these defense mechanisms also likely play an important role in neuroprotection.     

Repair of oxidative DNA damage in Drosophila melanogaster: identification and characterization of dOgg1, a second DNA glycosylase activity for 8-hydroxyguanine and formamidopyrimidines

In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using γ-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from γ-irradiated DNA. with k_cat/K_M values of 21.0 × 10^–5 and 11.2 × 10^–5 (min^–1 nM^–1), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a β-elimination reaction at the 3′-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.     

Reduction of 8-hydroxyguanine in human leukocyte DNA by physical exercise

We investigated the effect of physical exercise on the level of 8-hydroxyguanine (8-OH-Gua), a form of oxidative DNA damage, and its repair activity in human peripheral leukocytes. Whole blood samples were collected by venipuncture from 21 healthy male volunteers (10 trained athletes and 13 untrained men), aged 19-50 years, both before and after physical exercise. Trained athletes showed a lower level of 8-OH-Gua (2.4 ± 0.5/10⁶ Gua, p = 0.0032) before exercise when compared to that of untrained men (6.2 ± 3.5). The mean levels of 8-OH-Gua of untrained subjects decreased significantly (p = 0.0057) from 6.2 ± 3.5/10⁶ Gua (mean ± SD/10⁶ Gua) to 3.3 ± 1.4/10⁶ Gua after physical exercise. On the other hand, the mean levels of repair activity of untrained subjects significantly increased after exercise (p = 0.0093) from 0.037 ± 0.024 (mean DNA cleavage ratio ± SD) to 0.056 ± 0.036. In the trained athletes 8-OH-Gua level and its repair activity were not changed before and after the exercise. We also observed inter-individual differences in 8-OH-Gua levels and its repair activities. These results suggest that physical exercise causes both rapid and long-range reduction of oxidative DNA damage in human leukocytes, with individually different efficiencies.     

Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease

Mitochondrial generation of reactive oxygen species (ROS) is increased in mice with fatty livers induced by genetic obesity, chronic consumption of ethanol, or methionine/choline-deficient diets. Both nuclear and mitochondrial (mt) DNA are targets for ROS-induced damage and accumulate hydroxylated bases, such as 8-hydroxy-2'-deoxyguanosine (8-oxoG) and base substitution of adenine with 8-oxoG (A*8-oxoG), that introduce mutations that promote cancer as well as cell death. The mammalian homolog of the bacterial DNA mismatch repair enzyme MutY (MYH) removes A*8-oxoG from nuclear and mtDNA, reduces 8-oxoG accumulation, and restores genomic stability after ROS exposure. Cumulative damage to mtDNA occurs as fatty liver disease progresses. Therefore, differences in hepatic MYH activity may influence the severity of fatty liver disease. To evaluate this hypothesis, we compared mtH2O2 production, MYH expression, oxidative DNA damage, and hepatocyte death in healthy mice and different mouse models of fatty liver disease. The results show that diverse causes of steatohepatitis increase mtROS production, limit repair of mtDNA, and oxidatively damage DNA. However, there are important differences in the DNA repair response to oxidant stress among mouse models of fatty liver disease. Independent of the degree of mtROS generation, models with the least MYH exhibit the greatest accumulation of 8-oxoG and the most hepatocyte death. These findings raise the intriguing possibility that inherited or acquired differences in DNA repair enzyme activity may underlie some of the interindividual differences in fatty liver disease outcomes.     

The role of mammalian NEIL1 protein in the repair of 8-oxo-7,8-dihydroadenine in DNA

8-oxo-7,8-dihydroadenine (8-oxoAde) is a major product of adenine modification by reactive oxygen species. So far, only one mammalian DNA glycosylase, 8-oxoguanine-DNA-glycosylase 1 (OGG1), has been shown to excise 8-oxoAde, exclusively from pairs with Cyt. We have found that endonuclease VIII-like protein 1 (NEIL1), a mammalian homolog of bacterial endonuclease VIII, can efficiently remove 8-oxoAde from 8-oxoAde:Cyt pairs but not from other contexts. In an in vitro reconstituted system, reactions containing OGG1 produced a fully repaired product, whereas NEIL1 caused an abortive initiation of repair, stopping after 8-oxoAde removal and DNA strand cleavage. This block was partially relieved by polynucleotide kinase/3′-phosphatase. Thus, two alternative routes of 8-oxoAde repair may exist in mammals.     

Acetylation of human 8-oxoguanine-DNA glycosylase by p300 and its role in 8-oxoguanine repair in vivo

The human 8-oxoguanine-DNA glycosylase 1 (OGG1) is the major DNA glycosylase responsible for repair of 7,8-dihydro-8-oxoguanine (8-oxoG) and ring-opened fapyguanine, critical mutagenic DNA lesions that are induced by reactive oxygen species. Here we show that OGG1 is acetylated by p300 in vivo predominantly at Lys338/Lys341. About 20% of OGG1 is present in acetylated form in HeLa cells. Acetylation significantly increases OGG1's activity in vitro in the presence of AP-endonuclease by reducing its affinity for the abasic (AP) site product. The enhanced rate of repair of 8-oxoG in the genome by wild-type OGG1 but not the K338R/K341R mutant, ectopically expressed in oxidatively stressed OGG1-null mouse embryonic fibroblasts, suggests that acetylation increases OGG1 activity in vivo. At the same time, acetylation of OGG1 was increased by about 2.5-fold after oxidative stress with no change at the polypeptide level. OGG1 interacts with class I histone deacetylases, which may be responsible for its deacetylation. Based on these results, we propose a novel regulatory function of OGG1 acetylation in repair of its substrates in oxidatively stressed cells.     

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.