Metallothionein-III prevents gamma-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.     

Immunofluorescent detection of 8-oxoguanine DNA lesions in liver cells from aging OXYS rats, a strain prone to overproduction of free radicals

Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.     

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.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site.

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

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.     

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.     

A DNA aptamer sensor for 8-oxo-7,8-dihydroguanine

Abasic site-containing DNA duplex is a versatile structural motif that can be used for the design of purine aptamers and sensors. In this study, several modifications were introduced to the abasic site to explore possible specific binding of free 8-oxoG, a product of DNA base excision repair. The nucleoside opposite the abasic site was replaced by pyrrolo-dC as a reporter group. Binding of 8-oxoG quenched pyrrolo-dC fluorescence by as much as 70%. In contrast, adenine, guanine, thymine, and cytosine showed only minimal fluorescence quenching effect. The best aptamer binds 8-oxoG with a dissociation constant of 5.5±0.8μM. This sensor can be used to accurately measure 8-oxoG concentrations in the presence of guanine.     

Coordinated action of the Fanconi anemia and ataxia telangiectasia pathways in response to oxidative damage

Fanconi anemia (FA) and ataxia telangiectasia (AT) share common traits such chromosomal instability and proneness to hematological cancers. Both AT and FA cell lines, and patients, are characterized by abnormally high levels of oxidative stress markers. The key FA protein FANCD2 is phosphorylated on Ser 222 by ATM after ionizing radiation (IR), thus allowing normal activation of the S-phase checkpoint, and ATM cells are known to be hypersensitive to oxidative damage. In this work we show that FANCD2 deficient cells have a defective S-phase checkpoint after Hydrogen Peroxide (H(2)O(2)) induced oxidative damage. ATM dependent phosphorylation of FANCD2 at the S222 residue is necessary for normal S-phase checkpoint activation after oxidative stress, while FANCD2 monoubiquitination at K561 is dispensable. We also show that FANCD2 is not required for base excision repair of 8-oxoG and other DNA lesions (abasic sites, uracils), while treatments that exclusively induce 8-oxoG, but not DNA double strand breaks, fail to activate FANCD2 monoubiquitination, thus indicating that the known accumulation of 8-oxoG in FA cells reflects an overproduction of ROS rather than defective processing of oxidized bases. We conclude that the handling of DNA damage after H(2)O(2)-induced oxidative stress requires the coordinated action of FANCD2 and ATM.     

Product inhibition and magnesium modulate the dual reaction mode of hOgg1

8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3' to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.     

Structural Characterization of Clostridium acetobutylicum 8-Oxoguanine DNA Glycosylase in Its Apo Form and in Complex with 8-Oxodeoxyguanosine

DNA is subject to a multitude of oxidative damages generated by oxidizing agents from metabolism and exogenous sources and by ionizing radiation. Guanine is particularly vulnerable to oxidation, and the most common oxidative product 8-oxoguanine (8-oxoG) is the most prevalent lesion observed in DNA molecules. 8-OxoG can form a normal Watson-Crick pair with cytosine (8-oxoG:C), but it can also form a stable Hoogsteen pair with adenine (8-oxoG:A), leading to a G:C → T:A transversion after replication. Fortunately, 8-oxoG is recognized and excised by either of two DNA glycosylases of the base excision repair pathway: formamidopyrimidine-DNA glycosylase and 8-oxoguanine DNA glycosylase (Ogg). While Clostridium acetobutylicum Ogg (CacOgg) DNA glycosylase can specifically recognize and remove 8-oxoG, it displays little preference for the base opposite the lesion, which is unusual for a member of the Ogg1 family. This work describes the crystal structures of CacOgg in its apo form and in complex with 8-oxo-2′-deoxyguanosine. A structural comparison between the apo form and the liganded form of the enzyme reveals a structural reorganization of the C-terminal domain upon binding of 8-oxoG, similar to that reported for human OGG1. A structural comparison of CacOgg with human OGG1, in complex with 8-oxoG containing DNA, provides a structural rationale for the lack of opposite base specificity displayed by CacOgg.     

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.     

Structural basis for the lack of opposite base specificity of Clostridium acetobutylicum 8-oxoguanine DNA glycosylase

7,8-Dihydro-8-oxoguanine (8-oxoG) is the major oxidative product of guanine and the most prevalent base lesion observed in DNA molecules. Because 8-oxoG has the capability to form a Hoogsteen pair with adenine (8-oxoG:A) in addition to a normal Watson–Crick pair with cytosine (8-oxoG:C), this lesion can lead to a G:C → T:A transversion after replication. However, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Members of the Ogg1 family usually display a strong preference for a C opposite the lesion. In contrast, the atypical Ogg1 from Clostridium actetobutylicum (CacOgg) can excise 8-oxoG when paired with either one of the four bases, albeit with a preference for C and A. Here we describe the first high-resolution crystal structures of CacOgg in complex with duplex DNA containing the 8-oxoG lesion paired to cytosine and to adenine. A structural comparison with human OGG1 provides a rationale for the lack of opposite base specificity displayed by the bacterial Ogg.     

Efficiency of excision of 8-oxo-guanine within DNA clustered damage by XRS5 nuclear extracts and purified human OGG1 protein

A major DNA lesion is the strongly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) base, formed by oxidative attack at guanine and which leads to a high level of G.C-->T.A transversions. Clustered DNA damages are formed in DNA following exposure to ionizing radiation or radiomimetic anticancer agents and are thought to be biologically severe. The presence of 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell, if the OGG1 DNA glycosylase/AP lyase protein, present in eukaryotic cells, does not efficiently excise its substrate, 8-oxoG. In this study, specific oligonucleotide constructs containing an 8-oxoG located in several positions opposite to another damage (5,6-dihydrothymine (DHT), uracil, 8-oxoG, AP site, or various types of single strand breaks) were used to determine the relative efficiency of purified human OGG1 and mammalian XRS5 nuclear extracts to excise 8-oxoG from clustered damages. A base damage (DHT, uracil, and 8-oxoG) on the opposite strand has little or no influence on the rate of excision of 8-oxoG whereas the presence of either an AP site or various types of single strand breaks has a strong inhibitory effect on the formation of a SSB due to the excision of 8-oxoG by both hOGG1 and the nuclear extract. The binding of hOGG1 to 8-oxoG is not significantly affected by the presence of a neighboring lesion.     

The C-terminal Lysine of Ogg2 DNA Glycosylases is a Major Molecular Determinant for Guanine/8-Oxoguanine Distinction

7,8-Dihydro-8-oxoguanine (8-oxoG) is a major oxidative lesion found in DNA. The 8-oxoguanine DNA glycosylases (Ogg) responsible for the removal of 8-oxoG are divided into three families Ogg1, Ogg2 and AGOG. The Ogg2 members are devoid of the recognition loop used by Ogg1 to discriminate between 8-oxoG and guanine and it was unclear until recently how Ogg2 enzymes recognize the oxidized base. We present here the first crystallographic structure of an Ogg2 member, Methanocaldococcus janischii Ogg, in complex with a DNA duplex containing the 8-oxoG lesion. This structure highlights the crucial role of the C-terminal lysine, strictly conserved in Ogg2, in the recognition of 8-oxoG. The structure also reveals that Ogg2 undergoes a conformational change upon DNA binding similar to that observed in Ogg1 glycosylases. Furthermore, this work provides a structural rationale for the lack of opposite base specificity in this family of enzymes.     

Different DNA repair strategies to combat the threat from 8-oxoguanine

Oxidative DNA damage is one of the most common threats to genome stability and DNA repair enzymes provide protection from the effects of oxidized DNA bases. In mammalian cells, base excision repair (BER) mediated by the OGG1 and MYH DNA glycosylases prevents the accumulation of 8-oxoguanine (8-oxoG) in DNA. When steady-state levels of DNA 8-oxoG were measured in myh(-/-) and myh(-/-)/ogg1(-/-) mice, an age-dependent accumulation of the oxidized purine was found in lung and small intestine of doubly defective myh(-/-)/ogg1(-/-) mice. Since there is an increased incidence of lung and small intestinal cancer in myh(-/-)/ogg1(-/-) mice, these findings are consistent with a causal role for unrepaired oxidized DNA bases in cancer development. We previously presented in vitro evidence that mismatch repair (MMR) participates in the repair of oxidative DNA damage and msh2(-/-) mouse embryo fibroblasts also have increased steady state levels of DNA 8-oxoG. To investigate whether DNA 8-oxoG also accumulates in vivo, basal levels were measured in several organs of 4-month-old msh2(-/-) mice and their wild-type counterparts. Msh2(-/-) mice had significantly increased levels of DNA 8-oxoG in spleen, heart, liver, lung, kidney and possibly small intestine but not in bone marrow, thymus or brain. The tissue-specificity of DNA 8-oxoG accumulation in msh2(-/-) and other DNA repair defective mice suggests that DNA protection of different organs is mediated by different combinations of repair pathways.     

Oxidative stress impairs the repair of oxidative DNA base modifications in human skin fibroblasts and melanoma cells

Irradiation of mammalian cells with solar light is associated with the generation of reactive oxygen species (ROS) and oxidative stress, which is mediated in part by endogenous photosensitizers absorbing in the visible range of the solar spectrum. Accordingly, oxidative DNA base modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are the predominant types of DNA damage in cells irradiated at wavelengths >400 nm. We have analysed the repair of oxidative purine modifications in human skin fibroblasts and melanoma cells using an alkaline elution technique, both under normal conditions and after depletion of glutathione. Similar repair rates were observed in fibroblasts and melanoma cells from three different patients (t1/2 approximately 4h). In both cell types, glutathione depletion (increased oxidative stress) caused a pronounced repair retardation even under non-toxic irradiation conditions. Furthermore, the cleavage activity at 8-oxoG residues measured in protein extracts of the cells dropped transiently after irradiation. An addition of dithiothreitol restored normal repair rates. Interestingly, the repair rates of cyclobutane pyrimidine dimers (t1/2 approximately 18 h), AP sites (t1/2 approximately 1h) and single-strand breaks (t1/2 <30 min) were not affected by the light-induced oxidative stress. We conclude that the base excision repair of oxidative purine modifications is surprisingly vulnerable to oxidative stress, while the nucleotide excision repair of pyrimidine dimers is not.     

Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.     

MTH1, an oxidized purine nucleoside triphosphatase, prevents the cytotoxicity and neurotoxicity of oxidized purine nucleotides

In human and rodent cells, MTH1, an oxidized purine nucleoside triphosphatase, efficiently hydrolyzes oxidized dGTP, GTP, dATP and ATP such as 2'-deoxy-8-oxoguanosine triphosphate (8-oxo-dGTP) and 2'-deoxy-2-hydroxyadenosine triphosphate (2-OH-dATP) in nucleotide pools, thus avoiding their incorporation into DNA or RNA. MTH1 is expressed in postmitotic neurons as well as in proliferative tissues, and it is localized both in the mitochondria and nucleus, thus suggesting that MTH1 plays an important role in the prevention of the mutagenicity and cytotoxicity of such oxidized purines as 8-oxoG which are known to accumulate in the cellular genome. Our recent studies with MTH1-deficient mice or cells revealed that MTH1 efficiently minimizes accumulation of 8-oxoG in both nuclear and mitochondrial DNA in the mouse brain as well as in cultured cells, thus contributing to the protection of the brain from oxidative stress.     

APE1-dependent repair of DNA single-strand breaks containing 3'-end 8-oxoguanine

DNA single-strand breaks containing 3′-8-oxoguanine (3′-8-oxoG) ends can arise as a consequence of ionizing radiation and as a result of DNA polymerase infidelity by misincorporation of 8-oxodGMP. In this study we examined the mechanism of repair of 3′-8-oxoG within a single-strand break using purified base excision repair enzymes and human whole cell extracts. We find that 3′-8-oxoG inhibits ligation by DNA ligase IIIα or DNA ligase I, inhibits extension by DNA polymerase β and that the lesion is resistant to excision by DNA glycosylases involved in the repair of oxidative lesions in human cells. However, we find that purified human AP-endonuclease 1 (APE1) is able to remove 3′-8-oxoG lesions. By fractionation of human whole cell extracts and immunoprecipitation of fractions containing 3′-8-oxoG excision activity, we further demonstrate that APE1 is the major activity involved in the repair of 3′-8-oxoG lesions in human cells and finally we reconstituted repair of the 3′-8-oxoG-containing oligonucleotide duplex with purified human enzymes including APE1, DNA polymerase β and DNA ligase IIIα.     

Replication-associated repair of adenine:8-oxoguanine mispairs by MYH

Cellular DNA is constantly exposed to the risk of oxidation. 8-oxoguanine (8-oxoG) is one of the major DNA lesions generated by oxidation, which is primarily corrected by base excision repair. When it is not repaired prior to replication, replicative DNA polymerases yield misinsertion of an adenine (A) opposite the 8-oxoG on the template strand, generating an A:8-oxoG mispair. MYH, a mammalian homolog of Escherichia coli MutY, is a DNA glycosylase responsible for initiating base excision repair of such a mispair by excising the adenine opposite 8-oxoG. Here, using an in vivo repair system, we show that DNA replication enhances the repair of the A:8-oxoG mispair. Repair efficiency was lower in MYH-deficient murine cells than in MYH-proficient cells. Transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:8-oxoG repair, indicating that a significant part of this replication-associated repair depends on MYH. Expression of a mutant MYH in which the PCNA binding motif was disrupted did not increase the repair efficiency, thus suggesting that the interaction between PCNA and MYH is critical for MYH-initiated repair of A:8-oxoG.