A novel assay of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity in cultured cells and its use for evaluation of cadmium(II) inhibition of this activity.

8-Oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a product of oxidative modification of dGTP, thatcan be misincorporated into DNA, causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTP-ases) that convert it to 8-oxo-dGMP. Thus, inhibition of 8-oxo-dGTPases may lead to cancer. To elucidate the involvement of 8-oxo-dGTPases in carcinogenesis, an assay of the 8-oxo-dGTPase activity is required. This paper presents such an assay developed for Chinese hamster ovary (CHO) cells that can be applied to any biological material. It includes: (i) a convenient method for preparing 8-oxo-2'-deoxyguanosine 5'-phosphates; (ii) an HPLC/UV quantification of 8-oxo-dGTP hydrolysis products and (iii) separation of 8-oxo-dGTPase activity from interfering 8-oxo-dGTP phosphatase(s). The 8-oxo-dGTPase activity of CHO cells depends on magnesium, has a pH optimum of 8.5, Km for 8-oxo-dGTP of 9.3 microM, and is inhibited by 8-oxo-dGDP, the product of interfering 8-oxo-dGTP phosphatases. The latter must be removed from the assayed samples by ultrafiltration through 30 kDa cut-off membranes. The method was used to test the inhibition by cadmium ions of the activity of 8-oxo-dGTPase in CHO cells. The cells cultured with 0.3-3 microM cadmium(II) acetate for up to 24 h had their 8-oxo-dGTPase activity suppressed in a Cd(II) concentration-dependent manner, down to 70% of the control value.     

An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.     

CiMutT, an asidian MutT homologue, has a 7, 8-dihydro-8-oxo-dGTP pyrophosphohydrolase activity responsible for sanitization of oxidized nucleotides in Ciona intestinalis

The oxidized nucleotide precursors 7, 8-dihydro-8-oxo-dGTP (8-oxo-dGTP) and 1, 2-dihydro-2-oxo-dATP (2-oxo-dATP) are readily incorporated into nascent DNA strands during replication, which would cause base substitution mutations. E. coli MutT and human homologue hMTH1 hydrolyze 8-oxo-dGTP, thereby preventing mutations. In this study, we searched for hMTH1 homologues in the ascidian Ciona intestinalis using the NCBI-BLAST database. Among several candidates, we focused on one open reading frame, designated as CiMutT, because of its high degree of identity (41.7%) and similarity (58.3%) to the overall amino acid sequence of hMTH1, including the Nudix box. CiMutT significantly suppressed the mutator activity of E. coli mutT mutant. Purified CiMutT had a pyrophosphohydrolase activity that hydrolyzed 8-oxo-dGTP to 8-oxo-dGMP and inorganic pyrophosphate. It had a pH optimum of 9.5 and Mg(++) requirement with optimal activity at 5 mM. The activity of CiMutT for 8-oxo-dGTP was comparable to that of hMTH1, while it was 100-fold lower for 2-oxo-dATP than that of hMTH1. These facts indicate that CiMutT is a functional homologue of E. coli MutT. In addition, the enzyme hydrolyzed all four of the unoxidized nucleoside triphosphates, with a preference for dATP. The specific activity for 8-oxo-dGTP was greater than that for unoxidized dATP and dGTP. These results suggest that CiMutT has the potential to prevent mutations by 8-oxo-dGTP in C. intestinalis.     

Assessing the metabolic function of the MutT 8-oxodeoxyguanosine triphosphatase in Escherichia coli by nucleotide pool analysis

In Escherichia coli the mutT gene is one of several that acts to minimize mutagenesis by reactive oxygen species. The bacterial MutT protein and its mammalian homolog have been shown to catalyze in vitro the hydrolysis of the oxidized deoxyguanosine nucleotide, 8-oxo-dGTP, to its corresponding monophosphate. Thus, the protein is thought to "sanitize" the nucleotide pool by ridding the cell of a nucleotide whose incorporation into DNA would be intensely mutagenic. However, because others have shown mutT mutations to be mutagenic under some conditions of anaerobic growth, and have shown 8-oxo-dGTP to be a poor DNA polymerase substrate, there is reason to question this model. We have devised an assay for 8-oxo-dGTP in bacterial extracts. Using this assay, which involves reversed-phase high-performance liquid chromatography and electrochemical detection, we have been unable to detect 8-oxo-dGTP in extracts of three different mutT mutants of E. coli, even after growth of the bacteria in the presence of hydrogen peroxide. Our estimated upper limit for 8-oxo-dGTP content of these bacteria is about 200 molecules/cell, corresponding to a concentration of about 0.34 microm. When 8-oxo-dGTP was added at 0.34 microm to an in vitro DNA replication system primed with a DNA template that permits scoring of replication errors and with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect upon the frequency of replication errors. These findings lead us to question the conclusion that 8-oxo-dGTP is the most significant physiological substrate for the MutT protein.     

Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala⁵¹⁰ and Asn⁵¹³, play differential roles in dNTP selectivity. Specifically, Ala⁵¹⁰ and Asn⁵¹³ facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.     

NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5'-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans

8-oxo-dGTP is generated in the nucleotide pool by direct oxidation of dGTP or phosphorylation of 8-oxo-dGDP. It can be incorporated into DNA during replication, which would result in mutagenic consequences. The frequency of spontaneous mutations remains low in cells owing to the action of enzymes degrading such mutagenic substrates. Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP to 8-oxo-dGMP. Human NUDT5 as well as human MTH1 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP. These enzymes prevent mutations caused by misincorporation of 8-oxo-dGTP into DNA. In this study, we identified a novel MutT homolog (NDX-1) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. NDX-1 did not hydrolyze 8-oxo-dGTP, 2-hydroxy-dATP or 2-hydroxy-dADP. Expression of NDX-1 significantly reduced spontaneous A:T to C:G transversions and mitigated the sensitivity to a superoxide-generating agent, methyl viologen, in an E. coli mutT mutant. In C. elegans, RNAi of ndx-1 did not affect the lifespan of the worm. However, the sensitivity to methyl viologen and menadione bisulfite of the ndx-1-RNAi worms was enhanced compared with that of the control worms. These facts indicate that NDX-1 is involved in sanitization of 8-oxo-dGDP and plays a critical role in defense against oxidative stress in C. elegans.     

Expression of human MutT homologue (hMTH1) protein in primary non-small-cell lung carcinomas and histologically normal surrounding tissue

In situ, oxidation of deoxyguanosine yields 8-hydroxy-2'-deoxyguanosine (8-oxo-dG), which is mutation prone and results in a G:C --> T:A transversion following DNA replication. Another pathway to the formation of DNA containing 8-oxo-dG is by the misincorporation of 8-oxo-dGTP via DNA polymerase. Human MutT homologue (hMTH1), an 8-oxo-dGTPase, prevents misincorporation of this oxidized nucleotide by hydrolyzing 8-oxo-dGTP to 8-oxo-dGMP. Previous studies have shown that hMTH1 mRNA is overexpressed in human renal cell carcinomas and breast tumors. Elevated levels of hMTH1 protein have also been detected in brain tumors. In the current study, we determined whether hMTH1 protein is overexpressed in primary non-small-cell lung carcinomas as compared to adjacent histologically normal lung tissue. Twenty matched human lung tumor/normal pairs were examined by Western analysis for expression of hMTH1 protein. Overexpression in the tumors was detected in 4/8 (50%) adenocarcinomas, 4/4 (100%) adenocarcinomas with bronchioalveolar (BAC) features, 2/2 (100%) BACs, and 3/6 (50%) squamous cell carcinomas. The data from Western analysis were validated by immunohistochemical staining for hMTH1 protein. The results of this study indicate that hMTH1 protein may be a potential marker for the detection of persistent oxidative stress in lung cancer.     

Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.     

Inhibition of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity of the antimutagenic human MTH1 protein by nucleoside 5'-diphosphates

The hMTH1 protein, a human homologue of E. coli MutT protein, is an enzyme converting 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate. It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As found in our previous investigations, 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1. Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line). Among them, the strongest inhibitors appeared to be dGDP (Ki=74 microM), dADP (Ki=147 microM), and GDP (Ki=502 microM). Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range. Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 microM and GDP at mean concentration of 30 microM, taken together, can decrease the cellular hMTH1 enzymatic activity vs. 8-oxo-dGTP (expected to remain below 500 pM) by up to 15%. The other five NDPs and dNDPs tested cannot markedly affect this activity.     

Effects of base excision repair proteins on mutagenesis by 8-oxo-7,8-dihydroguanine (8-hydroxyguanine) paired with cytosine and adenine

8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2′-deoxyguanosine 5′-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C → T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T → C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T → C:G mutations caused by 8-oxo-dGTP.     

Critical amino acids in human DNA polymerases η and κ involved in erroneous incorporation of oxidized nucleotides

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Polη and Polκ proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Polη was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Polη) to 7 : 1 (R61K). Similarly, Tyr112 in Polκ was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polκ) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Polη, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Polη and Polκ might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.     

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.     

Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity

Replication of the mitochondrial genome by DNA polymerase γ requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from ~1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase γ. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3′ exonuclease activity of pol γ. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease.     

Functional significance of the conserved residues for the 23-residue module among MTH1 and MutT family proteins

Human MTH1 and Escherichia coli MutT proteins hydrolyze 7, 8-dihydro-8-oxo-dGTP (8-oxo-dGTP) to monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine into nascent DNA. Although only 30 amino acid residues (23%) are identical between MTH1 and MutT, there is a highly conserved region consisting of 23 residues (MTH1, Gly(36)-Gly(58)) with 14 identical residues. A chimeric protein MTH1-Ec, in which the 23-residue sequence of MTH1 was replaced with that of MutT, retains its capability to hydrolyze 8-oxo-dGTP, thereby indicating that the 23-residue sequences of MTH1 and MutT are functionally and structurally equivalent and constitute functional modules. By saturation mutagenesis of the module in MTH1, 14 of the 23 residues proved to be essential to exert 8-oxo-dGTPase activity. For the other 9 residues (40, 42, 44, 46, 47, 49, 50, 54, and 58), positive mutants were obtained, and Arg(50) can be replaced with hydrophobic residues (Val, Leu, or Ile), with a greater stability and higher specific activity of the enzyme. Indispensabilities of Val(39), Ile(45), and Leu(53) indicate that an amphipathic property of alpha-helix I consisting of 14 residues of the module (Thr(44)-Gly(58)) is essential to maintain the stable catalytic surface for 8-oxo-dGTPase.     

Cleavage of oxidized guanine nucleotide and ADP sugar by human NUDT5 protein

MutT-related proteins, including Escherichia coli MutT and the human MTH1 (NUDT1), degrade 8-oxo-7, 8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and thereby prevent mutations caused by the misincorporation of 8-oxoguanine into DNA. The human NUDT5, which has an intrinsic activity to cleave ADP sugars to AMP and sugar phosphate, possesses the ability to degrade 8-oxo-dGDP to the monophosphate. Since 8-oxo-dGDP and 8-oxo-dGTP are interconvertible by cellular enzymes, NUDT5 has the potential to prevent errors during DNA replication. The two activities associated with NUDT5 exhibit different pH dependencies; the optimum for the cleavage of ADP ribose is pH 7-9, while that for 8-oxo-dGDPase is around pH 10. The kinetic parameters for the two types of reactions indicated that ADP ribose is a better substrate for NUDT5 compared with oxidized guanine nucleotides. The 8-oxo-dGDP cleavage was competitively inhibited by ADP ribose and its reaction product, AMP, and in reverse, the cleavage of ADP ribose was inhibited by 8-oxo-dGDP. These results imply that the two types of substrates may share the same binding site for catalysis.     

A Serendipitous Synthesis of 8-Dimsyl-2′-deoxyguanosine

Abstract

A serendipitous synthesis of 8-dimsyl-dG (2) has been achieved along with the known 8-benzyloxy-dG (3) in a nucleophilic substitution reaction of 8-bromo-dG (1) with in situ generated dimsyl and benzyloxy sodium. Compound 3 was directly converted into the mutagenic oxidative DNA damage product, 8-oxo-dGTP (4).     

An oxidized purine nucleoside triphosphatase,MTH1, suppresses cell death caused by oxidative stress

MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA. In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria. The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases. A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H2O2. All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity. Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction.