8-Chloro-dGTP, a hypochlorous acid-modified nucleotide, is hydrolyzed by hMTH1, the human MutT homolog

The human mutT homolog, hMTH1, suppresses spontaneous mutations by degrading the endogeneous mutagen, 8-hydroxy-dGTP. We previously reported the broad substrate specificity of hMTH1, which also degrades the oxidatively damaged purine nucleotides, 2-hydroxy-dATP, 8-hydroxy-dATP, 2-hydroxy-ATP, and 8-hydroxy-GTP, in addition to 8-hydroxy-dGTP. In this paper, we describe the hMTH1 activity for 8-chloro-dGTP, which could be formed in inflamed tissue by the reaction of dGTP with hypochlorous acid, a product of myeloperoxidase from activated human neutrophils. The hMTH1 protein was mixed with 1-20 microM of 8-chloro-dGTP and 8-hydroxy-dGTP, and the reaction products were quantified by anion-exchange HPLC to measure the pyrophosphatase reaction rate. The kinetic parameters revealed that 8-chloro-dGTP was degraded by hMTH1 with 50% efficiency as compared with that of 8-hydroxy-dGTP. This result suggests that 8-chloro-dGTP is an intrinsic substrate for hMTH1.     

Probing the substrate recognition mechanism of the human MTH1 protein by nucleotide analogs

To examine the substrate recognition mechanism of the human MTH1 protein, which hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxy-dGTP, ten nucleotide analogs (8-bromo-dATP, 8-bromo-dGTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the MTH1 protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. The fact that the syn-oriented brominated nucleotides were hydrolyzed suggests that the MTH1 protein binds to deoxynucleotides adopting the syn-conformation. However, 8-hydroxy-dITP, which lacks the 2-amino group of 8-hydroxy-dGTP, was degraded with tenfold less efficiency as compared with 8-hydroxy-dGTP. In addition, deoxyisoinosine triphosphate, lacking the 6-amino group of 2-hydroxy-dATP, was hydrolyzed as efficiently as 8-hydroxy-dGTP, but less efficiently than 2-hydroxy-dATP. These results clarify the effects of the anti/syn conformation and the functional groups on the 2 and 6 positions of the purine ring on the recognition by the human MTH1 protein.     

Recognition of nucleotide analogs containing the 7,8-dihydro-8-oxo structure by the human MTH1 protein

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.     

The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein.

The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5'-cyclo-dATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.     

Important amino acids in the phosphohydrolase module of Escherichia coli Orf135

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. In this study, the Gly-36, Gly-37, Lys-38, Glu-43, Arg-51, Glu-52, Leu-53, Glu-55, and Glu-56 residues of Orf135, which are conserved in the three MutT-type proteins (Orf135, MutT, and MTH1), were substituted, and the enzymatic activity of these mutant proteins was examined. The mutant proteins with a substitution at the 36th, 37th, 52nd, and 56th amino acid residues completely lost their activity. On the other hand, the mutant proteins with a substitution at the 38th, 43rd, 51st, 53rd, and 55th residues could hydrolyze 5-methyl-dCTP. Some mutants with detectable activity for 5-methyl-dCTP did not hydrolyze dCTP. Activities for known substrates (5-methyl-dCTP, dCTP, 2-hydroxy-dATP, and 8-hydroxy-dGTP) were examined in detail with the four mutants, K38R, E43A, L53A, and E55Q. These results indicate the essential residues for the activity of the Orf135 protein.     

Molecular genetics and structural biology of human MutT homolog, MTH1

The human MTH1 gene located on chromosome 7p22 consists of 5 major exons. MTH1 gene produces seven types of mRNAs and the B-type mRNAs with exon 2b-2c segments direct synthesis of three forms of MTH1 polypeptides (p22, p21, and p18) by alternative initiation of translation, while the others encode only p18. In human cells, p18, the major form is mostly localized in the cytoplasm with some in the mitochondria. A single nucleotide polymorphism (SNP) in exon 2, which is tightly liked to another SNP (GTG83/ATG83), creates an additional alternative in-frame AUG in B-type MTH1 mRNAs yielding the fourth MTH1 polypeptide, p26 that possesses an additional mitochondrial targeting signal. These SNPs are likely to be one of the risk factors for cancer or for neuronal degeneration. The 30 amino acid residues are identical between MTH1 and MutT, and there is a highly conserved region consisting of 23 residues (MTH1: Gly36 to Gly58), with 14 identical residues. A chimeric protein in which the 23 residue sequence of MTH1 was replaced with that of MutT, retains the capability to hydrolyze 8-oxo-dGTP, indicating that the 23 residue sequences of MTH1 and MutT are functionally and structurally equivalent, and constitute a functional phosphohydrolase module. Saturated mutagenesis of the module in MTH1 indicated that an amphipathic property of the alpha-helix I consisting of 14 residues of the module (Thr44 to Gly58) is essential to maintain the stable catalytic surface for 8-oxo-dGTPase. MTH1 but not MutT efficiently hydrolyzes two forms of oxidized dATP, 2-hydroxy-dATP and 8-oxo-dATP, as well as 8-oxo-dGTP and 8-oxo-GTP. Thus, MTH1 is designated as the oxidized purine nucleoside triphosphatase and has a much wider substrate specificity than MutT. There is a significant homology between MTH1 protein and the C-terminal half of human MYH protein, which may be involved in the recognition of 8-oxoguanine and 2-hydroxyadenine.     

Structure of human MTH1, a Nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates

Oxygen radicals generated through normal cellular respiration processes can cause mutations in genomic and mitochondrial DNA. Human MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP and 2-hydroxy-dATP, to monophosphates, thereby preventing the misincorporation of these oxidized nucleotides during replication. Here we present the solution structure of MTH1 solved by multidimensional heteronuclear NMR spectroscopy. The protein adopts a fold similar to that of Escherichia coli MutT, despite the low sequence similarity between these proteins outside the conserved Nudix motif. The substrate-binding pocket of MTH1, deduced from chemical shift perturbation experiments, is located at essentially the same position as in MutT; however, a pocket-forming helix is largely displaced in MTH1 (approximately 9 A) such that the shape of the pocket differs between the two proteins. Detailed analysis of the pocket-forming residues enabled us to identify Asn33 as one of the key residues in MTH1 for discriminating the oxidized form of purine, and mutation of this residue modifies the substrate specificity. We also show that MTH1 catalyzes hydrolysis of 8-oxo-dGTP through nucleophilic substitution of water at the beta-phosphate.     

Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.     

Base excision repair enzyme endonuclease III suppresses mutagenesis caused by 8-hydroxy-dGTP

To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) and 2-hydroxy-dATP were introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei) and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. The mutations induced by exogenous 2-hydroxy-dATP were similar in all of the strains tested. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 2-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.     

Role of P2 Receptors as Modulators of Rat Eosinophil Recruitment in Allergic Inflammation

ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step was to investigate the expression and functionality of the P2X receptors by patch clamping, our results showed a potency ranking order of ATP>ATPγS> 2meSATP> ADP> αβmeATP> βγmeATP>BzATP> UTP> UDP>cAMP. This data suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency (UTP>ATPγS> ATP > UDP> ADP >2meSATP > αβmeATP) suggests the presence of P2Y2, P2Y4, P2Y6 and P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did chemotaxis assays to verify whether nucleotides could induce migration. After 1 or 2 hours of incubation, ATP increased migration of eosinophils, as well as ATPγS, a less hydrolysable analogue of ATP, while suramin a P2 blocker abolished migration. In keeping with this idea, we tested whether these receptors are implicated in the migration of eosinophils to an inflammation site in vivo, using a model of rat allergic pleurisy. In fact, migration of eosinophils has increased when ATP or ATPγS were applied in the pleural cavity, and once more suramin blocked this effect. We have demonstrated that rat eosinophils express P2X and P2Y receptors. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo, an effect blocked by suramin.     

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.     

Purinergic receptor stimulation increases membrane trafficking in brown adipocytes

Stimulation of brown adipocytes by their sympathetic innervation plays a major role in body energy homeostasis by regulating the energy- wasting activity of the tissue. The norepinephrine released by sympathetic activity acts on adrenergic receptors to activate a variety of metabolic and membrane responses. Since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. We find that micromolar concentrations of extracellular ATP initiates profound changes in the membrane trafficking of brown adipocytes. ATP elicited substantial increases in total cell membrane capacitance, averaging approximately 30% over basal levels and occurring on a time scale of seconds to minutes. The membrane capacitance increase showed an agonist sensitivity of 2-methylthio-ATP > or = ATP > ADP > > adenosine, consistent with mediation by a P2r type purinergic receptor. Membrane capacitance increases were not seen when cytosolic calcium was increased by adrenergic stimulation, and capacitance responses to ATP were similar in the presence and absence of extracellular calcium. These results indicate that increases in cytosolic calcium alone do not mediate the membrane response to ATP. Photometric assessment of surface-accessible membrane using the dye FM1- 43 showed that ATP caused an approximate doubling of the amount of membrane actively trafficking with the cell surface. The discrepancy in the magnitudes of the capacitance and fluorescence changes suggests that ATP both activates exocytosis and alters other aspects of membrane handling. These findings suggest that secretion, mobilization of membrane transporters, and/or surface membrane expression of receptors may be regulated in brown adipocytes by P2r purinergic receptor activity.     

Transient contraction of muscle fibers on photorelease of ATP at intermediate concentrations of Ca2+.

We isometrically activated skinned fibers in rigor by flash photolysis of caged ATP at various [Ca2+] at 8 degrees C. On release of ATP, tension initially decreased with the same time course at all [Ca2+]. At high [Ca2+] (pCa < or = 5.8), tension rose to the steady-state plateau after the brief relaxation. When the [Ca2+] was intermediate (7.0 < or = pCa < or = 6.0), tension temporarily overshot the final steady-state level. The half-time during this tension transient was longer at higher [Ca2+]. The transient contractions could be simulated by a simple kinetic model: R + ATP-->Q, and X<-->Q<-->A, where R, X, and A are the rigor, relaxed, and active-tension states, respectively; Q is a "pre-active" state where tension is very low; and Ca2+ affects only the X-Q transition. This scheme was also useful for predicting the tension transients in Ca(2+)- and P(i)-jump experiments at various [Ca2+]. ADP enhanced the Ca2+ sensitivity of the ATP-induced transient contraction, which was not in the scope of the model.     

Increased A:T-->C:G mutations in the mutT strain upon 8-hydroxy-dGTP treatment: direct evidence for MutT involvement in the prevention of mutations by oxidized dGTP

The Escherichia coli MutT protein hydrolyzes 8-hydroxy-dGTP (8-OH-dGTP) in vitro, and mutT gene deficiencies cause increased spontaneous A:T-->C:G mutations. However, no direct evidence exists for enhanced mutagenicity of 8-OH-dGTP in mutT cells. In this study, 8-OH-dGTP was introduced into wild type and mutT E. coli cells, and mutations of a chromosomal gene were monitored. 8-OH-dGTP induced mutations of the rpoB gene, the degree of the mutation induction in the mutT strain being approximately 6-fold higher than that in the wild type strain. On the other hand, 2-hydroxy-dATP, which is not a substrate of the MutT protein, increased the mutation to similar degrees in the two strains. These results constitute the first evidence that the MutT protein suppresses mutation by 8-OH-dGTP in vivo.     

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-- >E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s- 1.     

Cystic Fibrosis Transmembrane Conductance Regulator–associated ATP Release Is Controlled by a Chloride Sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl⁻ that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl⁻ conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl⁻ conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl⁻ binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl⁻. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation.

Phosphorylated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels require nucleoside triphosphates, such as ATP, to open. As the concentration of intracellular ATP increases, the probability of the channel being open (Po) increases. To better understand how ATP regulates the channel, we studied excised inside-out membrane patches that contained single, phosphorylated CFTR Cl- channels and examined the kinetics of gating at different concentrations of ATP. As the ATP concentration increased from 0.1 to 3 mM the mean closed time decreased, but mean open time did not change. Analysis of the data using histograms of open- and closed-state durations, the maximum likelihood method, and the log-likelihood ratio test suggested that channel behavior could be described by a model containing one open and two closed states (C1<==>C2<==>O). ATP regulated phosphorylated channels at the transition between the closed states C1 and C2: as the concentration of ATP increased, the rate of transition from C1 to C2 (C1-->C2) increased. In contrast, transitions from C2 to C1 and between C2 and the open state (O) were not significantly altered by ATP. Addition of ADP in the presence of ATP decreased the transition rate from C1 to C2 without affecting other transition rates. These data suggest that ATP regulates CFTR Cl- channels through an interaction that increases the rate of transition from the closed state to a bursting state in which the channel flickers back and forth between an open and a closed state (C2). This transition may reflect ATP binding or perhaps a step subsequent to binding.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

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.