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.     

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress

The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents β-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes.     

Spontaneous loss-of-function mutations of the 8-oxoguanine DNA glycosylase gene in mice and exploration of the possible implication of the gene in senescence

8-Oxoguanine is one of the major premutagenic oxidative base legions in vivo and is suspected to play a crucial role in various pathophysiological processes, such as cancer and aging. Mammalian 8-oxoguanine DNA glycosylase (OGG1) is thought to play a major role in the removal of 8-oxoguanine adducts in vivo. We have identified several inbred mouse strains with a spontaneous mutation, OGG1-R336H or double mutations, OGG1-R304W/R336H. R304W mutation caused a complete loss of OGG1 activity, while the R336H mutation led to disruption of nuclear localization of the enzyme although the activity remained normal. Among the double mutants was SAMP1, which exhibits accelerated senescence and short lifespan. We assessed the possible implication of the mutant OGG1 and 8-oxoguanine in aging utilizing SAMP1 mice. SAMP1 retained 1.5- to 1.9-fold increase in 8-oxoguanine level of hepatic nuclear DNA as compared with normal mice, until at least 12 months of age. A genetic association study, however, indicated that the mutant Ogg1 gene per se is not responsible for the accelerated senescence and short lifespan of SAMP1. Mutant OGG1 may be associated with pathologic conditions in other mouse strains.     

Cardiac overexpression of 8-oxoguanine DNA glycosylase 1 protects mitochondrial DNA and reduces cardiac fibrosis following transaortic constriction

Cardiac failure is associated with increased levels of oxidized DNA, especially mitochondrial (mtDNA). It is not known if oxidized mtDNA contributes to cardiac dysfunction. To test if protection of mtDNA can reduce cardiac injury, we produced transgenic mice with cardiomyocyte-specific overexpression of the DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) isoform 2a. In one line of mice, the transgene increased OGG1 activity by 115% in mitochondria and by 28% in nuclei. OGG1 transgenic mice demonstrated significantly lower cardiac mitochondrial levels of the DNA guanine oxidation product 7,8-dihydro-8-oxoguanine (8-oxo-dG) under basal conditions, after doxorubicin administration, or after transaortic constriction (TAC), but the transgene produced no detectable reduction in nuclear 8-oxo-dG content. OGG1 mice were tested for protection from the cardiac effects of TAC 13 wk after surgery. Compared with FVB-TAC mice, hearts from OGG1-TAC mice had lower levels of β-myosin heavy chain mRNA but they did not display significant differences in the ratio of heart weight to tibia length or protection of cardiac function measured by echocardiography. The principle benefit of OGG1 overexpression was a significant decrease in TAC-induced cardiac fibrosis. This protection was indicated by reduced Sirius red staining on OGG1 cardiac sections and by significantly decreased induction of collagen 1 and 3 mRNA expression in OGG1 hearts after TAC surgery. These results provide a new model to assess the damaging cardiac effects of 8-oxo-dG formation and suggest that increased repair of 8-oxo-dG in mtDNA decreases cardiac pathology.     

Phosphorylation of human oxoguanine DNA glycosylase (alpha-OGG1) modulates its function

Oxoguanine DNA glycosylase (OGG1) initiates the repair of 8-oxoguanine (8-oxoG), a major oxidative DNA base modification that has been directly implicated in cancer and aging. OGG1 functions in the base excision repair pathway, for which a molecular hand-off mechanism has been proposed. To date, only one functional and a few physical protein interactions have been reported for OGG1. Using the yeast two-hybrid system and a protein array membrane, we identified two novel protein interactions of OGG1, with two different protein kinases: Cdk4, a serine-threonine kinase, and c-Abl, a tyrosine kinase. We confirmed these interactions in vitro using recombinant proteins and in vivo by co-immunoprecipitation from whole cell extracts. OGG1 is phosphorylated in vitro by Cdk4, resulting in a 2.5-fold increase in the 8-oxoG/C incision activity of OGG1. C-Abl tyrosine phosphorylates OGG1 in vitro; however, this phosphorylation event does not affect OGG1 8-oxoG/C incision activity. These results provide the first evidence that a post-translational modification of OGG1 can affect its catalytic activity. The distinct functional outcomes from serine/threonine or tyrosine phosphorylation may indicate that activation of different signal transduction pathways modulate OGG1 activity in different ways.     

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.     

DNA repair gene 8-oxoguanine DNA glycosylase Ser326Cys polymorphism and colorectal cancer risk in a Kashmiri population

8-Oxoguanine DNA glycosylase (OGG1) is one of the important base excision repair enzymes that repair 8-oxoguanine lesion incorporated within the DNA of an individual by reactive oxygen species. The aim of this study was to detect the role of OGG1 Ser326Cys polymorphism in susceptibility to colorectal cancer (CRC) in a Kashmiri population. We investigated the genotype distribution of the OGG1 gene in 114 CRC cases in comparison with 200 healthy subjects. There was no significant association between OGG1 Ser326Cys polymorphism and CRC, but the homozygous Cys/Cys variant genotype was associated with an increased risk of colon cancer (p<0.05). This study suggests that the OGG1 polymorphism is not associated with the risk of development of CRC in the Kashmiri population in general but modulates the risk of cancer development in colon via interaction with many dietary factors.     

PARP-1 Expression is Increased in Colon Adenoma and Carcinoma and Correlates with OGG1

The ethiology of colon cancer is largely dependent on inflammation driven oxidative stress. The analysis of 8-oxodeoxyguanosine (8-oxodGuo) level in leukocyte DNA of healthy controls (138 individuals), patients with benign adenomas (AD, 137 individuals) and with malignant carcinomas (CRC, 169 individuals) revealed a significant increase in the level of 8-oxodGuo in leukocyte DNA of AD and CRC patients in comparison to controls. The counteracting mechanism is base excision repair, in which OGG1 and PARP-1 play a key role. We investigated the level of PARP-1 and OGG1 mRNA and protein in diseased and marginal, normal tissues taken from AD and CRC patients and in leukocytes taken from the patients as well as from healthy subjects. In colon tumors the PARP-1 mRNA level was higher than in unaffected colon tissue and in polyp tissues. A high positive correlation was found between PARP-1 and OGG1 mRNA levels in all investigated tissues. This suggests reciprocal influence of PARP-1 and OGG1 on their expression and stability, and may contribute to progression of colon cancer. PARP-1 and OGG1 proteins level was several fold higher in polyps and CRC in comparison to normal colon tissues. Individuals bearing the Cys326Cys genotype of OGG1 were characterized by higher PARP-1 protein level in diseased tissues than the Ser326Cys and Ser326Ser genotypes. Aforementioned result may suggest that the diseased cells with polymorphic OGG1 recruit more PARP protein, which is necessary to remove 8-oxodGuo. Thus, patients with decreased activity of OGG1/polymorphism of the OGG1 gene and higher 8-oxodGuo level may be more susceptible to treatment with PARP-1 inhibitors.     

Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

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.     

Balb/c小鼠增龄过程中不同脏器线粒体DNA损伤及损伤修复相关基因的变化

为探讨Balb/c小鼠增龄过程中线粒体DNA损伤及其修复基因表达与衰老之间的关系,采用逆转录 多聚酶链反应（RT PCR）方法,检测年轻与老年Balb/c小鼠脑、肝脏和脾脏中线粒体自身编码基因细胞色素氧化酶亚单位Ⅰ基因（coⅠ）和细胞色素氧化酶亚单位Ⅲ基因（coⅢ）及8 氧鸟嘌呤糖基化酶基因（ogg1）、DNA聚合酶γ（DNA polymeraseγ）基因、胸腺嘧啶乙二醇DNA糖基化酶基因（nth1）等碱基切除修复基因在mRNA水平的变化.用Western印迹方法检测小鼠脾脏中COⅢ和OGG1的蛋白质水平的变化.结果发现,老年小鼠脾脏中coⅠ和coⅢ的mRNA水平比年轻小鼠显著增加（P<0.05）,CO Ⅲ的蛋白质水平亦比年轻小鼠显著升高（P<0.05）;老年小鼠脾脏OGG1的mRNA和蛋白水平上均比年轻小鼠显著增加（P<0.05）.老年小鼠肝脏和脾脏DNA聚合酶γ和NTH1的mRNA水平比年轻小鼠显著升高（P<0.05）.提示线粒体DNA自身编码的基因及碱基切除修复基因的表达失衡可能是Balb/c小鼠衰老的原因之     

Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases

The eukaryotic 8-oxoguanine-DNA glycosylase 1 (OGG1) provides the major activity for repairing mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) induced in the genome due to oxidative stress. Earlier in vitro studies showed that, after excising the base lesion, the human OGG1 remains bound to the resulting abasic (AP) site in DNA and does not turn over efficiently. The human AP-endonuclease (APE1), which cleaves the phosphodiester bond 5' to the AP site, in the next step of repair, displaces the bound OGG1 and thus increases its turnover. Here we show that NEIL1, a DNA glycosylase/AP lyase specific for many oxidized bases but with weak 8-oxoG excision activity, stimulates turnover of OGG1 in a fashion similar to that of APE1 and carries out betadelta-elimination at the AP site. This novel collaboration of two DNA glycosylases, which do not stably interact with each other, in stimulating 8-oxoguanine repair is possible because of higher AP site affinity and stronger AP lyase activity of NEIL1 relative to OGG1. Comparable levels of NEIL1 and OGG1 in some human cells raise the possibility that NEIL1 serves as a backup enzyme to APE1 in stimulating 8-oxoG repair in vivo.     

Effects of melanin and manganese on DNA damage and repair in PC12-derived neurons

The mechanism of neurotoxicity produced by the interaction of melanin with manganese was investigated in PC12-derived neuronal cell cultures. The cells were incubated with melanin (25-500 microg/ml), MnCl2 (10 ng/ml-100 microg/ml) and a combination of both substances for 24 and 72 h. Incubation with either toxicant alone resulted in a minimal decrease in cell viability. The combination of melanin and manganese caused significant (up to 60%) decreases in viability of PC12 cells in a dose-dependent manner. Increases in oxidative DNA damage, indicated by levels of 8-hydroxy-2'deoxyguanosine (8-oxodG), was associated with decreased cell viability. Melanin alone, but not manganese alone, resulted in increased oxidative DNA damage. The maximal increase in 8-oxodG caused by melanin was about seven times higher than control after 24 h of exposure. The activity of the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), was increased in cells incubated with single toxicants and their combinations for 24 h. On the third day of incubation with the toxicants, activity of OGG1 declined below control levels and cell viability significantly decreased. Melanin was observed to have an inhibitory effect on OGG1 activity. Study of the regulation of OGG1 activity in response to melanin and manganese may provide insights into the vulnerability of nigral neurons to oxidative stress in Parkinson's disease.     

Thermolabile 8-hydroxyguanine DNA glycosylase with low activity in senescence-accelerated mice due to a single-base mutation.

8-hydroxyguanine (8-oxoguanine; oh8Gua) DNA glycosylase (OGG1) repairs oh8Gua, a highly mutagenic oxidative DNA damage. In the present study, we compared two strains of senescence-accelerated mouse (SAM) expressing senescence-prone phenotypes, SAMP1 and SAMP8, with one strain of SAM expressing senescence-resistant phenotype, SAMR1. We found three distinct characteristics of OGG1 in SAMPs: (i) low activity (10-40% of the SAMRI enzyme in all organs and ages observed), (ii) thermolability, and (iii) mutation from Arg (CGG) in SAMR1 to Trp (TGG) at codon 304. There was no difference in the levels of mRNA and protein. As expected, oh8Gua level in tissues was higher in the SAMPs. In contrast, O6-methylguanine-DNA methyltransferase, which repairs alkylated DNA, showed no difference in its activity. The impairment of oh8Gua repair activity caused by the 304 mutation in OGG1 may be one of the factors contributing to the high somatic mutation rate and the accelerated senescence observed in these strains.     

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.     

Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells

Reactive oxygen species threaten genomic integrity by inducing oxidative DNA damage. One common form of oxidative DNA damage is the mutagenic lesion 8-oxoguanine (8-oxodG). One driver of oxidative stress that can induce 8-oxodG is inflammation, which can be initiated by the cytokine tumor necrosis factor alpha (TNF-α). Oxidative DNA damage is primarily repaired by the base excision repair pathway, initiated by glycosylases targeting specific DNA lesions. 8-oxodG is excised by 8-oxoguanine glycosylase 1 (OGG1). A common Ogg1 allelic variant is S326C-Ogg1, prevalent in Asian and Caucasian populations. S326C-Ogg1 is associated with various forms of cancer, and is inactivated by oxidation. However, whether oxidative stress caused by inflammatory cytokines compromises OGG1 variant repair activity remains unknown. We addressed whether TNF-α causes oxidative stress that both induces DNA damage and inactivates S326C-OGG1 via cysteine 326 oxidation. In mouse embryonic fibroblasts, we found that S326C-OGG1 was inactivated only after exposure to H₂O₂ or TNF-α. Treatment with the antioxidant N-acetylcysteine prior to oxidative stress rescued S326C-OGG1 activity, demonstrated by in vitro and cellular repair assays. In contrast, S326C-OGG1 activity was unaffected by potassium bromate, which induces oxidative DNA damage without causing oxidative stress, and presumably cysteine oxidation. This study reveals that Cys326 is vulnerable to oxidation that inactivates S326C-OGG1. Physiologically relevant levels of TNF-α simultaneously induce 8-oxodG and inactivate S326C-OGG1. These results suggest a mechanism that could contribute to increased risk of cancer among S326C-Ogg1 homozygous individuals.