Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties.

A cDNA encoding murine O6-alkylguanine-DNA-alkyltransferase (ATase) has been sequenced after isolation from total liver RNA by the polymerase chain reaction using oligonucleotide primers derived from the rat ATase cDNA sequence. Functionally active murine ATase protein has been expressed in Escherichia coli at high levels (about 2% of total protein) and purified to apparent homogeneity (molecular mass 26 kDa). In liquid hybridization experiments, anti-human ATase polyclonal antibodies inhibited human but not rat or mouse ATase, whereas anti-rat polyclonal antibodies inhibited rat and mouse but not human ATase. Both antibodies detected all mammalian ATases tested by western analysis so far. These results indicate some common epitopes and at least one unique human epitope. We compared the amino-acid sequence of the murine ATase with those of other mammalian and bacterial ATases. The proteins of this family all have a large domain (approximately 70 amino acids) of highly conserved residues flanking the sequence PCHRV, which contains the alkyl-accepting cysteine residue of the active site. No evidence was found in the sequences for helix-turn-helix, leucine-zipper, or zinc-finger motifs for DNA recognition and binding. Nuclear localization signals (basic-residue-rich regions) could not be uniquely identified in the mammalian members of the family. Outside of the conserved PCHRV region, there were major differences between prokaryotic and eukaryotic proteins at the primary structure level: there was a series of proline-rich motifs, but these also varied between sequences.     

ogt alkyltransferase enhances dibromoalkane mutagenicity in excision repair–deficient escherichia coli K-12

We examined the role of the O⁶-alkylguanine-DNA alkyltransferase encoded by ogt gene in the sensitivity of Escherichia coli to the mutagenic effects of the dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt^? bacteria to those in their isogenic ogt⁺ parental counterparts. The effects of the uvrABC excision-repair system, the adaptive response, mucAB and umuDC mutagenic processing, and glutathione bioactivation on the differential responses of ogt^? and ogt⁺ bacteria were also studied. Mutation induction was monitored by measuring the frequency of forward mutations to L -arabinose resistance. Induced mutations occurred only in excision repair–defective strains and were totally (with dibromomethane) or substantially (with dibromoethane) dependent on the alkyltransferase (ATase) encoded by the ogt gene. An increased mutagenic response to both dibromoalkanes was also seen in ogt^? bacteria that overexpressed the ogt protein from a multicopy plasmid, indicating that the differences in mutability between ogt⁺ and ogt^? bacteria were not dependent on the ogt^? null allele carried by the defective strain. The ATase encoded by the constitutive ogt gene was more effective in promoting dibromoalkane mutagenicity than the ada ATase induced by exposure to low doses of a methylating agent. The mutagenicity promoted by the ogt ATase was dependent on both glutathione bioactivation and SOS mutagenic processing. To our knowledge, this paper presents for the first time evidence that DNA ATases, in particular the ATase encoded by the ogt gene, can increase the mutagenic effects of a DNA-damaging agent. The mechanism of this effect has yet to be established. © 1995 Wiley-Liss Inc.     

Comparison of the inactivation of mammalian and bacterial O6-alkylguanine-DNA alkyltransferases by O6-benzylguanine and O6-methylguanine.

The inactivation of human and Escherichia coli O6-alkylguanine-DNA alkyltransferase by O6-methylguanine and O6-benzylguanine was compared. When HT29 cell extracts or E. coli Ada protein were incubated in the presence of 200 microM O6-methylguanine for 1 h, alkyltransferase activity was reduced to 44 and 39% of control levels respectively. However, under the same conditions O6-benzylguanine completely depleted alkyltransferase activity in the extract from human cells but had virtually no effect on the Ada protein. Incubation of the HT29 cell alkyltransferase with O6-benzyl[3H]guanine resulted in a time-dependent production of [3H]guanine. No similar production of [3H]guanine was observed in the presence of the Ada protein. In CHO cells transfected with the bacterial ada gene (CHO-ada) or the human alkyltransferase cDNA (CHO-MGMT), treatment with 500 microM O6-methylguanine inhibited both alkyltransferases by greater than 85%. In contrast, 2 microM O6-benzylguanine inhibited human alkyltransferase expressed in CHO-MGMT cells by greater than 99% though concentrations as high as 25 microM for 24 h had no inhibitory effects on the bacterial alkyltransferase expressed in CHO-ada cells. This selective inhibition was also observed in vivo in transgenic mice expressing ada in the liver where O6-benzylguanine caused a decrease of only 40% in total hepatic alkyltransferase activity compared to 95% in non-transgenic mice, consistent with inhibition of only the mammalian alkyltransferase and maintenance of bacterial alkyltransferase activity in these animals. Thus, while O6-methylguanine at high concentrations inactivates both bacterial and mammalian alkyltransferases, O6-benzylguanine is a substrate only for the mammalian protein and is unable, perhaps due to steric hindrance, to inhibit the Ada protein.     

Variability and regulation of O6-alkylguanine-DNA alkyltransferase

O(6)-Alkylguanine-DNA alkyltransferase (ATase) confers resistance to many of the biological effects of certain classes of alkylating agents by repairing the DNA lesions responsible. The role of ATase in the mutagenic and toxic effects of the carcinogenic and antitumour alkylating agents are of interest in relation to the prevention and treatment of cancer in man. In this commentary we specifically focus on the variation in ATase levels and our current understanding of the factors involved in the regulation of ATase expression.     

Contribution of ogt-encoded alkyltransferase to resistance to chloroethylnitrosoureas in nucleotide excision repair-deficient Escherichia coli

We investigated the relative contribution of the two Escherichiacoli DNA alkyltransferases (ATases) to the increased sensitivityof ATase-deficient bacteria to the mutagenic and lethal effectsof chloroethylnitrosoureas (CNU). The ogt-encoded protein wasthe principal determinant in resistance to the mutagenic effectsof CNU in E.coli. Thus, only when the ogt gene was inactivatedwas sensitivity to mutagenesis greatly increased; the contributionof inactivation of the ada gene was relatively minor. Furthermore,induction of the adaptive response provided essentially no protectionagainst CNU mutagenesis in either an ogt⁺ or ogt     

Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas.

V79 Chinese hamster cells expressing either the O6-alkylguanine-DNA-alkyltransferase (ATase) encoded by the E. coli ogt gene or a truncated version of the E. coli ada gene have been exposed to various alkylnitrosoureas to investigate the contribution of ATase repairable lesions to the toxicity of these compounds. Both ATases are able to repair O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) but the ogt ATase is more efficient in the repair of O4-methylthymine (O4-MeT) and higher alkyl derivatives of O6-AlkG than is the ada ATase. Expression of the ogt ATase provided greater protection against the toxic effects of the alkylating agents then the ada ATase particularly with N-ethyl-N-nitrosourea (ENU) and N-butyl-N-nitrosourea (BNU) to which the ada ATase expressing cells were as sensitive as parent vector transfected cells. Although ogt was expressed at slightly higher levels than the truncated ada in the transfected cells, this could not account for the differential protection observed. For-N-methyl-N-nitrosourea (MNU) the increased protection in ogt-transfected cells is consistent with O4-MeT acting as a toxic lesion. For the longer chain alkylating agents and chloroethylating agents, the protection afforded by the ogt protein may be a consequence of the more efficient repair of O6-AlkG, O4-AlkT or both of these lesions in comparison with the ada-encoded ATase.     

Enhanced repair of O6-methylguanine DNA adducts in the liver of transgenic mice expressing the ada gene

The capacity to repair O6-methylguanine-DNA adducts was measured in the liver of transgenic mice expressing a chimeric gene consisting of the inducible P-enolpyruvate carboxykinase (GTP) promoter linked to the bacterial O6-alkylguanine-DNA alkyltransferase (ada) gene. Under induced conditions, total hepatic alkyltransferase reached 32.8 +/- 4.2 (SE) fmol/micrograms DNA compared to 7.8 +/- 1.1 fmol/micrograms DNA in nontransgenic mice. Administration of methylnitrosourea or nitrosodimethylamine to both groups of mice produced O6-methylguanine-DNA adducts which resulted in repair-mediated depletion of total hepatic alkyltransferase in a dose-dependent fashion. In nontransgenic mice, depletion of hepatic alkyltransferase occurred at lower doses of carcinogen, and recovery of alkyltransferase activity occurred later than in ada+ transgenic mice. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of residual alkyltransferase activity after methylating agent exposure indicated that the bacterial as well as endogenous mammalian alkyltransferases were functioning as DNA repair proteins in hepatocytes in vivo. Analysis of O6-methylguanine- and N7-methylguanine-DNA adducts in the liver of transgenic and nontransgenic mice after treatment with one dose of 50 mg/kg methylnitrosourea i.p. revealed that transgenic mice repaired in situ O6-methylguanine-DNA adducts approximately 3 times faster than nontransgenic mice, commensurate with the increase in alkyltransferase activity. Thus, ada+ transgenic mice treated with methylnitrosourea have lower levels of persistent mutagenic O6-methylguanine adducts than ada- nontransgenic mice. Hepatic expression of bacterial alkyltransferase appears to protect mice from the DNA-damaging effects of N-nitroso compounds in vivo.     

Expression in mammalian cells of a truncated Escherichia coli gene coding for O6-alkylguanine alkyltransferase reduces the toxic effects of alkylating agents

The lesion O⁶-alkylguanine (O⁶-AG) is produced in cellular DNAfollowing exposure to monofunctional alkylating agents and itsmiscoding and mutagenic properties have been demonstrated inspecific in vitro systems. In order to examine whether thislesion could be responsible for any of the biological effectsof alkylating agents in mammalian cells, we have constructeda plasmid containing the O⁶-AG alkyltransferase (ATase) regionof the gene from Escherichia coli, the product of which normallyrepairs both O⁶-AG and alkyl-phosphotriesters in DNA. We havetransfected the construction into Chinese hamster fibroblastswhich are deficient in endogenous ATase activity and selecteda clone that expresses the truncated repair gene. We demonstratethat this protein is functional, acts on damage in host cellDNA and protects the cells from the toxic effects of those alkylatingagents that react extensively at oxygen atom positions.     

Low O6-alkylguanine DNA-alkyltransferase activity in normal colorectal tissue is associated with colorectal tumours containing a GC-->AT transition in the K-ras oncogene

O6-alkylguanine DNA-alkyltransferase (ATase) provides protection against the toxic, mutagenic and carcinogenic effects of alkylating agents, principally by removing the promutagenic lesion O6-alkylguanine from DNA. Differences in ATase activity in human tissue may thus determine mutational susceptibility. As GC-->AT transitions, which can be induced by O6-alkylguanine in DNA, are commonly observed in the K- ras oncogene of alkylating agent induced animal tumours and in human colorectal tumours, we have examined whether differences in ATase activity may affect the risk of K-ras mutations in humans with colorectal tumours. NTase activity in normal tissue from individuals with a K-ras mutation in colorectal tissue and more specifically a GC-- >AT transition (but not a transversion mutation) was significantly lower than that in individuals without a mutation (P < 0.01). Thus, individuals with low ATase activity in normal tissue (i.e. below the median) were at increased risk of having a transition (OR 10.1; 95% CI 1.9-99.0), but not a transversion mutation (OR 1.7; 95% CI 0.3-12.2). There were no significant differences in tumour ATase activity in individuals with or without a mutation. These results suggest that ATase can protect colorectal tissue against the mutagenic effects of alkylating agents and furthermore, that alkylating agent exposure plays a role in the aetiology of colorectal tumours containing a GC-->AT transition in the K-ras oncogene.      

Improvement of chemotherapy efficacy by inactivation of a DNA-repair pathway

Tumour resistance and dose-limiting toxic effects restrict treatment with most chemotherapeutic drugs. Elucidation of the mechanisms of these effects could permit the development of ways to improve the effectiveness of currently used agents until better therapeutic agents are developed. Several types of alkylating agents are used in the treatment of cancer. The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (ATase) is an important cellular resistance mechanism to one class of alkylating agents. This enzyme removes potentially lethal damage from DNA and experiments in vitro and in vivo have shown that its inactivation can reverse resistance to such agents. Clinical trials of drugs that inactivate ATase are underway and early results indicate that they are active in tumour tissues. However, the ATase present in normal tissues, particularly bone marrow, is also inactivated, necessitating a reduction in the dose of alkylating agent. An important question is whether, in the absence of any tumour-specific delivery strategy, such drugs will improve therapeutic effectiveness; initial reports are not promising.     

Nitrosated peptides and polyamines as endogenous mutagens in O6- alkylguanine-DNA alkyltransferase deficient cells

Mutants of Escherichia coli and Saccharomyces cerevisiae that lack O6- alkylguanine-DNA alkyltransferase activities have increased spontaneous mutation rates, indicating the presence of a cellular metabolite that can alkylate DNA. Bacterially catalysed nitrosation has been implicated previously in producing the endogenous alkylating agent(s). Here, nitrosated polyamines and azaserine, a model compound for nitrosated peptides, are shown to be mutagenic to E. coli ada ogt mutants deficient in O6-alkylguanine-DNA alkyltransferase activity. The mutagenicity of azaserine may be explained by its ability to methylate DNA, whereas nitrosated spermidine causes DNA damage that is susceptible to both nucleotide excision repair and O6-alkylguanine-DNA alkyltransferase activity, which indicates the generation of more bulky DNA adducts. Nitrosated peptides and polyamines are therefore potential endogenous mutagens that are harmful particularly in O6-alkylguanine- DNA alkyltransferase deficient cells.      

Comparative aflatoxin B1 mutagenesis of Salmonella typhimurium TA 100 in metabolic and photoactivation systems

Salmonella typhimurium TA 100 was mutagenized with photoactivated aflatoxin B1 (AFB1) and AFB2. Levels of mutagenesis induced by AFB1 correlated with levels of in vitro covalent binding of [3H]AFB1 to calf thymus DNA. The same phenomenon was observed with AFB2. Photoactivated AFB1 induced lethality in the mutagenized cultures, and AFB2 failed to do so. Extraction of nucleic acids from cultures mutagenized by photoactivated or metabolically activated [3H]AFB1 revealed that: (a) in situ levels of [3H]AFB1 binding to DNA were proportional to induction of mutational and lethal events in both cases; (b) mammalian metabolism and photoactivation produced AFB1:DNA lesions possessing comparable lethality and mutagenicity; and (c) [3H]AFB1 binding levels to bacterial RNA did not correlate with mutagenesis and lethality.     

Isolation and cDNA cloning of a rat O6-alkylguanine-DNA-alkyltransferase gene, molecular analysis of expression in rat liver

A rat O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA has been isolated from a rat liver cDNA library by hybridization with the human homologue. The candidate 806 bp cDNA was sequenced and shown to contain a 630 bp open reading frame that could encode a protein of 22.2 kd. Fluorography of labelled ATase indicates a 24 kd protein which is consistent with several previous reports. The derived amino acid sequence demonstrated 81% similarity with the human ATase and 94% identity over a 67 residue region encompassing the putative alkyl acceptor site. Peptide sequences derived from cleaved homogeneous rat ATase have been located in the predicted protein providing additional confirmation of the identity of the cDNA. A 1.05 kb mRNA has been detected in rat liver by Northern analysis; treatment of adult rats with 2-acetylaminofluorene causes an approximately 10-fold induction of this message in liver. Following site directed mutagenesis of the 806 bp cDNA, the 630 bp protein coding sequence has been ligated into an Escherichia coli expression vector to achieve ATase levels of greater than 3% of total protein in bacterial extracts.     

Comparison of O6-alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissues

O⁶-Alkylguanine-DNA alkyltransferase (alkyltransferase) is therepair protein for O⁶-alkylguanine, a pre-mutagenic adduct formedby a variety of alkylating agents. Previous comparisons of therepair capacity of O⁶-alkylguanine in different tissues haveexpressed the alkyltransferase activity relative to total protein,and have asserted that tissues with low levels of activity wereat greater risk for mutagenic damage than tissues with higherlevels of activity. Because the alkyltransferase uses DNA assubstrate, and because tissues vary greatly in protein content,comparisons of tissue alkyltransferase activity may be moreappropriately based on cellular DNA content. We compared alkyltransferaseactivity relative to tissue DNA content with the activity relatedto protein content in human, rat and mouse tissues. In eachspecies, liver containing the highest level of activity usingeither method. In agreement with the findings of others, lowlevels of alkyltransferase activity relative to protein wereseen in human brain, rat brain and small intestine, and mousekidney. However, based on alkyltransferase activity relativeto DNA content, low levels of activity were seen in human bonemarrow myeloid precursors, rat bone marrow, brain and intestine,and mouse spleen and bone marrow. The range of activity betweentissues was 18-fold in human, 15-fold in rat and 8-fold in mouse.In general, the rank of alkyltransferase activity relative toDNA for each tissue was human > rat > mouse. These resultssuggest that the mouse is more susceptible to nitrosoureas thanrat or human. In each species, the organs with low levels ofalkyltransferase activity relative to tissue DNA content wouldappear to be targets for mutagenic damage following nitrosoureaexposure.     

O6-alkylguanine-DNA alkyltransferase depletion and regeneration in human peripheral lymphocytes following dacarbazine and fotemustine.

O6-Alkylguanine-DNA alkyltransferase (ATase) levels were measured in peripheral blood lymphocytes of 13 patients with advanced malignant melanoma treated with sequential dacarbazine (DTIC) and fotemustine. Wide interindividual variation in the pretreatment levels and in depletion and regeneration of ATase activity was noted. Depletion of ATase was seen within the first h after DTIC administration with values ranging from 44 to 92% of pretreatment levels. In 10 patients, progressive depletion of ATase activity occurred with nadir activity occurring at about 4 to 6 h with values ranging from 0 to 67% of pretreatment activity; at 18 h after DTIC infusion. ATase activity varied from 6 to 81%. No significant difference was seen between the rates of ATase depletion or regeneration between the two groups of patients receiving either 500 or 800 mg/m2 of DTIC with the same dose of fotemustine (100 mg/m2). In one patient, maximum depletion occurred within 1 h and no ATase activity was detectable over the next 18 h. In another patient, maximum depletion occurred at 2 h after DTIC followed by recovery of ATase activity to 71% at 18 h. In 2 patients who returned for subsequent cycles of chemotherapy, an increase in pretreatment ATase activity was seen. Overall, the extent of depletion of ATase following DTIC/fotemustine was directly proportional to the initial ATase level.     

In vivo depletion of O6-alkylguanine-DNA-alkyltransferase in lymphocytes and melanoma of patients treated with CB 10-277, a new DTIC analogue

Summary there is increasing evidence to suggest that alkylation of guanine residues in DNA at the O⁶ position is the critical cytotoxic event following treatment with dacarbazine (DTIC) and related drugs and that endogenous O⁶-alkylguanine-DNA alkyltransferase (ATase) gene expression may be a major factor in resistance to such agents. 1-p-Carboxyl-3,3-dimethylphenyltriazene (CB 10-277) was recently selected for clinical evaluation as a DTIC analogue with improved solubility, stability and (possibly) metabolic activation. Serial ATase levels were measured in peripheral blood lymphocytes of nine patients and in biopsied melanoma samples of two patients undergoing treatment with 24-h continuous infusion of CB 10-277 (12 g/m²). Wide individual variations in pre-treatment levels as well as in the post-treatment depletion of lymphocyte ATase were seen. Progressive depletion of lymphocyte ATase was seen during continuous infusion of CB 10-277 in all patients. Complete suppression of lymphocyte ATase activity occurred in two patients whose pre-treatment ATase levels were low. Immediately following completion of the CB 10-277 infusion, the median ATase activity was 17% of pre-treatment levels (range, 0–67%). At 24 h after the end of the infusion, no recovery of lymphocyte ATase activity was observed in six patients, but significant recovery to 50%, 100% and 102% of pre-treatment activity occurred in the other three. In three patients who returned for subsequent cycles of chemotherapy at 4 weeks after the first dose, pre-treatment ATase levels showed a 3-to 4-fold increase relative to the original pre-treatment values. A significant correlation was found between the extent of ATase depletion and the initial lymphocyte ATase levels (r=0.725,P<0.05). Haematological toxicity developed in two patients and was associated with low pre-treatment ATase activity. Depletion of tumour ATase activity was noted in these patients, with residual activity amounting to 8% and 11% of pre-treatment levels, respectively, in the biopsied melanoma tissues. These results indicate extensive metabolism of CB 10-277 to a methylating agent capable of mediating alkylation of DNA and subsequent depletion of lymphocyte and tumor ATase levels and further indicate that the effects on lymphocytes may reflect effects on the target tumour.Abbreviations ATase O⁶-alkylguanine-DNA alkyltransferase - CB 10-277 1-p-carboxyl-3,3-dimethylphenyltriazene - DTIC dacarbazine, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide - MTIC 5-(3-methyl-1-triazeno)imidazole-4-carboxamide     

DNA interstrand cross-linking and cytotoxicity induced by chloroethylnitrosoureas and cisplatin in human glioma cell lines which vary in cellular concentration of O6-alkylguanine-DNA alkyltransferase.

Fifteen human glioma cell lines were examined for their sensitivity to 1,3-bis(chloroethyl)-nitrosourea (BCNU, carmustine) and cis-dichlorodiamminoplatinum (cisplatin), the induction of DNA interstrand cross-linking (DNA-ISC) induced by the two agents and cellular O6-alkylguanine alkyltransferase (ATase) activity. Cell lines differed in their sensitivities to BCNU by up to 12-fold and to cisplatin by up to 21-fold. For both drugs, the extent of DNA-ISC was related to the drug sensitivity. There was a wide range of cellular ATase levels. Increasing ATase levels correlated with increased resistance to BCNU and with decreased formation of DNA-ISC following treatment with BCNU. In contrast, following treatment with cisplatin, there was no correlation between cellular ATase content and cytotoxicity or between ATase and DNA-ISC. Four sublines of varying ATase activity were prepared from one of the cell lines. These sublines showed a sensitivity to BCNU in inverse proportion to ATase activity, while sensitivity to cisplatin was more uniform. The experiments confirm the direct relationship between ATase concentration and sensitivity to BCNU in glioma cells. Although there was some correlation between cisplatin cytotoxicity and BCNU cytotoxicity, this was not mediated through ATase.     

Oxidation of methylhydrazines to mutagenic methylating derivatives and inducers of the adaptive response of Escherichia coli to alkylation damage.

The methylhydrazines, monomethylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine, are known carcinogens but only weak mutagens in the Ames test. Chemical oxidation of these compounds by potassium ferricyanide greatly enhanced their mutagenicity to an Escherichia coli ada mutant and converted them into inducers of the adaptive response of E. coli to alkylation damage. Enzymatic oxidation of monomethylhydrazine by horseradish peroxidase-H2O2 also yielded products which induced the adaptive response. Thus, methylhydrazines can be oxidized to active DNA-methylating derivatives which generate methylphosphotriesters (the inducing signal of the adaptive response), O6-methylguanine and/or O4-methylthymine (the miscoding bases repaired by the Ada protein) in DNA. These observations support the suggestion that metabolic oxidation of methylhydrazines in mammalian systems may be required to generate the mutagenic/carcinogenic derivatives.     

Inter- and intracellular heterogeneity of O6-alkylguanine-DNA alkyltransferase expression in human brain tumours: possible significance in nitrosourea therapy

The inter- and intracellular distribution of the DNA repairprotein O⁶-alkylguanine-DNA alkyltransferase (ATase) may bean important factor in the sensitivity or resistance of tumoursto treatment with certain alkylating agents, including the methyltriazenesand nitrosoureas. In order to examine this issue 26 human braintumour sections (23 high grade gliomas and three low grade gliomas)were examined for ATase expression by immunohistochemistry usingarabbit anti-human ATase polyclonal antibody. Positive staining,seen as fine black granules mainly confined to the nucleus,was observed in all the glioma sections examined. There wasmarked cellular heterogeneity, ranging from cells completelydevoidof staining to cells with very intense staining. Semi-quantitatively,in the 23 high grade gliomas examined six had1+ staining, sevenhad 2 + staining and 10 had 3 + staining, whereas all threelow grade gliomas had 1+ staining. These results are in contrastto published reports showing that     

Significance of error-avoiding mechanisms for oxidative DNA damage in carcinogenesis

Reactive oxygen species (ROS) are produced through normal cellular metabolism, and their formation is further enhanced by exposure to ionizing radiation and various chemicals. ROS attack DNA, and the resulting oxidative DNA damage is considered to contribute to aging, carcinogenesis and neurodegeneration. Among various types of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoguanine or 8-oxoG) is the most abundant, and plays significant roles in mutagenesis because of its ability to pair with adenine as well as cytosine. Enzymatic activities that may be responsible for preventing 8-oxoG-evoked mutations were identified in mammalian cells. We have focused on the following three enzymes: MTH1, OGG1 and MUTYH. MTH1 is a mammalian ortholog of Escherichia coli MutT, which hydrolyzes 8-oxo-dGTP to its monophosphate form in nucleotide pools, thereby preventing incorporation of the mutagenic substrate into DNA. OGG1, a functional counterpart of E. coli MutM, has an 8-oxoG DNA glycosylase activity. MUTYH, a mammalian ortholog of E. coli MutY, excises an adenine paired with 8-oxoG. These three enzymes are thought to prevent mutagenesis caused by 8-oxoG in mammals. To analyze the functions of mammalian MTH1 (Mth1), OGG1 (Ogg1) and MUTYH (Mutyh) in vivo, we established mutant mice for these three enzymes by targeted mutagenesis, and investigated spontaneous tumorigenesis as well as mutagenesis. Here we discuss our recent investigation of mutagenesis and carcinogenesis in these mutant mice.