Mouse MTH1 protein with 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphatase activity that prevents transversion mutation. cDNA cloning and tissue distribution.

8-Oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) is formed in the nucleotide pool of a cell during normal cellular metabolism, and when it is incorporated into DNA causes mutation. Organisms possess 8-oxo-dGTPase, an enzyme that specifically degrades 8-oxo-dGTP to 8-oxo-dGMP. We isolated cDNA for mouse 8-oxo-dGTPase, using as a probe human MTH1 (Escherichia coli mutT homolog) cDNA. The nucleotide sequence of the cDNA revealed that the mouse MTH1 protein (molecular weight of 17,896) comprises 156 amino acid residues. When the cDNA for mouse 8-oxo-dGTPase was expressed in E. coli mutT mutant cells devoid of their own 8-oxo-dGTPase activity, an 18-kDa protein, which is cross-reactive with an anti-human MTH1 antibody, was formed. In such cells, the level of spontaneous mutation frequency that was elevated reverted to normal. High levels of 8-oxo-dGTPase activity were found in liver, thymus, and large intestine, whereas all other organs examined contained smaller amounts of the enzyme. In embryonic stem cells, an exceedingly high level of the enzyme was present.

Oxygen radicals are generated during normal cellular metabolism, and the formation of such radicals is further enhanced by ionizing radiation and by various chemicals (1). Among many classes of DNA damage caused by oxygen radicals, an oxidized form of guanine base, 8-oxo-7,8-dihydroguanine (8-oxoguanine), 1 appears to play important roles in mutagenesis and in carcinogenesis (2)(3)(4). During DNA replication, 8-oxoguanine nucleotide can pair with cytosine and adenine nucleotides, with an almost equal efficiency, and transversion mutation ensues (5)(6)(7).
Organisms are equipped with elaborate mechanisms to counteract such mutagenic effects of 8-oxoguanine. In Escherichia coli, two glycosylases encoded by the mutM and the mutY genes function to prevent mutation caused by 8-oxoguanine in DNA (8 -12). MutM protein removes 8-oxoguanine paired with cyto-sine and MutY protein removes adenine paired with 8-oxoguanine. Enzyme activities similar to those of MutM and MutY have been identified in mammalian cells (13)(14)(15)(16). A significant amount of 8-oxoguanine is formed in the chromosomal DNA of mammalian cells, and most damaged nucleotides are excised from the DNA and excreted into the urine (17). These enzymes may maintain spontaneous mutation frequency at certain low levels.
Oxidation of guanine proceeds also in forms of free nucleotides, and an oxidized form of dGTP, 8-oxo-dGTP, is a potent mutagenic substrate for DNA synthesis (18). Organisms possess mechanisms for preventing mutation due to misincorporation of 8-oxo-dGTP. MutT protein of E. coli hydrolyzes 8-oxo-dGTP to the monophosphate, and lack of the mutT gene increases the occurrence of A:T to C:G transversion thousands of fold over the wild type level (18 -20). Human cells contain an enzyme similar to the MutT protein, and this enzyme specifically hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with a relatively low K m value, as compared with other deoxyribonucleoside triphosphates (21,22). Therefore, the human 8-oxo-dGTPase is likely to have the same antimutagenic capability as the MutT protein. Recently, the genomic sequence encoding the enzyme was isolated and the gene, named MTH1 (for mutT homolog), was seen to be located on human chromosome 7p22 (23).
To elucidate the roles of 8-oxo-dGTPase in carcinogenesis, it is necessary to construct animal models with altered levels of this enzyme activity, and it is of interest to determine whether the frequency of occurrence of tumors would increase in mice defective in the 8-oxo-dGTPase gene. As the first step toward this goal, we cloned cDNA for mouse MTH1 protein and characterized the product. We also investigated the distribution of the MTH1 protein in various organs of mice by quantitative immunoprecipitation and Western blot analysis, using an anti-MTH1 antibody.

EXPERIMENTAL PROCEDURES
Cells and Chemicals-The mouse embryonic stem cell line, CCE, was obtained from Dr. M. Katsuki, Kyushu University. C57BL/6J mice 8 Ϯ 1 weeks old, were obtained from CLEA, Inc. (Fukuoka, Japan). Plasmid pA1 containing mouse IAP elements was provided by Dr. Y. Nakatsu, Institute for Cancer Research, Fox Chase Cancer Center (Philadelphia, PA). [␣-32 P]dCTP, [␣-32 P]dGTP, and 125 I-labeled protein A were obtained from Amersham Japan (Tokyo). A DNA labeling kit was purchased from Nippon Gene (Toyama, Japan). Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and Klenow fragment were obtained from Toyobo Co. (Osaka, Japan). The cloning vector, pBluescript II KS ϩ , was purchased from Stratagene (La Jolla, CA), and the expression vector in E. coli, pTrc99A, was from Pharmacia Biotech Inc. (Uppsala, Sweden). RNA size standards and prestained protein molecular weight standards were obtained from Life Technologies, Inc. Rifampicin and protease inhibitors were obtained from Sigma, and IPTG was from Wako Pure Chemical (Osaka, Japan). Polyethyleneimine-cellulose plates were obtained from Merck. Activated CH-Sepharose 4B and protein A-Sepharose CL-4B were purchased from Pharmacia. Dialysis membranes (Spectra/Por membrane) and BIO-DOT, SF were obtained * This work was supported by grants from the Ministry of Education, Science and Culture of Japan and funding from the Human Frontier Science Program and from the Fukuoka Cancer Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Isolation of Mouse 8-Oxo-dGTPase cDNA Clones-A mouse cDNA library (a gift from Dr. D. E. Rancourt, University of Utah), prepared from an embryonic stem cell line, CC1.2, was screened by plaque hybridization using a random-primed NcoI/BamHI fragment (560 base pairs) excised from p24T that contained the entire coding region for human MTH1 cDNA (22). Prehybridization and hybridization were carried out at 42°C in solution containing 4 ϫ SSC (1 ϫ ϭ 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5 ϫ Denhardt's solution (1 ϫ ϭ 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 1% SDS, 40% formamide, and 100 g/ml heat-denatured salmon sperm DNA (Sigma). Hybridization transfer membranes (DuPont NEN) were washed twice at room temperature in 2 ϫ SSC, 0.1% SDS, and twice at 65°C in 0.5 ϫ SSC, 0.1% SDS, and then autoradiographed on Fuji RX filter at Ϫ80°C with an intensifying screen. Phage DNAs were isolated from clones showing positive signals and were checked for size of the inserted DNAs. The insert of clone M-2 which contains the 5Ј-most region of the cDNA, and those of three others, M-1, M-8, and M-12, were subcloned into pBluescript II KS ϩ for further analyses. The nucleotide sequences were determined using a model 373A automated DNA sequencer (Applied Biosystems/Perkin-Elmer Japan, Chiba, Japan).
Expression of Mouse cDNA in E. coli-A DNA fragment carrying the coding region for mouse MTH1 was amplified by reverse transcriptasepolymerase chain reacton with a set of primers designed from preceding sequences, 5Ј-CAGTCGACCATGGGCACCTCCAGGC-3Ј (Pm-1) and 5Ј-GTGTAGGATCCTGAGTGGCCAGAAC-3Ј (Pm-2), using the cDNA of poly(A) ϩ RNA from CCE cells as a template. The synthetic oligonucleotide primer (Pm-1) was designed to introduce the NcoI site at the start codon, which resulted in alteration of the second amino acid residue, serine, to glysine. A reverse transcriptase-polymerase chain reacton product of a 521-bp DNA fragment was cloned into the SmaI site of pBluescript II KS ϩ , and the nucleotide sequence of the amplified cDNA was confirmed by sequencing. After digestion with NcoI and BamHI, a 503-bp DNA fragment was excised and inserted into the NcoI/BamHI site of pTrc99A, producing pTK1.
A pair of isogenic E. coli strains, MK601 (mutT ϩ ) and MK602 (mutT Ϫ ), derived from AB1157 (12) were transformed with pTrc99A or pTK1 and grown overnight at 30°C in LB medium containing 50 g/ml ampicillin. The cultures were diluted with fresh medium and grown at 37°C until A 550 ϭ 0.6 -0.8. For induction, IPTG was added to give a final concentration of 1 mM, and incubation was extended for 6 h. From aliquots of the culture, the cells were collected and disrupted by sonication in lysis buffer (25 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 2 mM dithiothreitol) containing 0.5 mM phenylmethylsulfonyl fluoride, followed by centrifugation at 12,000 ϫ g at 4°C for 20 min. Aliquots of the lysate were used for analysis by SDS-polyacrylamide gel electrophoresis and for assay of 8-oxo-dGTPase activity.
Preparation of Anti-human MTH1 Antibody-Polyclonal rabbit antibodies against human MTH1 protein were prepared using TrpE human MTH1 fusion protein (24). The TrpE human MTH1 fusion protein was obtained as follows. The NarI (Klenow polished)/BamHI fragment from p24T (22) was subcloned into the PstI (Klenow polished)/BamHI site of pYN3103-TrpE vector (24). In this construct, the first and the second amino acid residues (Met and Gly) of the authentic human MTH1 were converted to Trp and Ala, respectively. The resulting NcoI/BamHI fragment of the plasmid, carrying the coding region for the TrpE human MTH1 fusion protein, was subcloned into the NcoI/BamHI site of the expression vector pET8c (25). The fusion protein was produced in E. coli BL21 and separated by SDS-polyacrylamide gel electrophoresis. A region of the gel containing the fusion protein (approximately 200 g) was excised to inoculate into the dorsum of a rabbit (Japanese white species) together with adjuvant (TiterMax, Hunter). Four weeks after the first immunization, the first booster (approximately 100 g) was given, and then boosters were given every other week. The immunoglobulin fraction was purified by ammonium sulfate precipitation, followed by affinity chromatography. Briefly, activated CH-Sepharose 4B was coupled with solubilized TrpE mouse MTH1 fusion protein (to prepare the TrpE fusion column), and with TrpE protein (for TrpE column) as ligands. Anti-TrpE antibodies were removed by passing through a TrpE column, and the flow-through fraction was applied onto the TrpE fusion column. Specific antibodies were obtained after elution from the column with a buffer at pH 2.3-2.5 and then dialyzed against Tris-buffered saline (10 mM Tris-HCl, pH 7.4, 0.9% NaCl). This antibody preparation was designated as anti-MTH1.
Assay of 8-Oxo-dGTPase Activity-The preparation was reacted in buffer containing 20 mM Tris-HCl, pH 8.0, 4 mM MgCl 2 , 40 mM NaCl, 20 M 8-oxo-dGTP, 80 g/ml BSA, 8 mM dithiothreitol, 2% glycerol, as described elsewhere (22). The reaction was run at 30°C for 20 min then was halted by applying 2 l of the reaction mixture onto a polyethyleneimine-cellulose sheet. 8-Oxo-dGMP produced was separated from 8-oxo-dGTP on TLC with 1 M LiCl for 90 min and quantitated in a Fujix 2000 Bio-image analyzer. One unit of 8-oxo-dGTPase activity was defined as the amount of enzyme that produced 1 pmol of 8-oxo-dGMP per min at 30°C (21).
Northern Blot Analysis-Total RNAs were isolated from mouse cell lines, NIH3T3 and CCE, and various tissues of mice, using the guanidinium thiocyanate/CsCl method (27). Poly(A) ϩ RNA was prepared from total RNA using Oligotex-dT 30 (Hoffmann-La Roche). Three g of poly(A) ϩ RNA from each cell line were applied to electrophoresis on a 1.2% agarose-formaldehyde gel, and the RNAs that were separated were transferred onto a nitrocellulose membrane (BA-85, Schleicher & Schuell) in 20 ϫ SSC by capillary blotting (28). The filter was hybridized using a 503-bp NcoI/BamHI fragment of mouse cDNA as a probe. Northern analysis of total RNAs (20 g) from various tissues of mice was done as described above, and the 18 S ribosomal RNA gene probe (29) obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) was used to quantify the amounts of RNA species on the blots. Filters were washed in 0.2 ϫ SSC and 0.2% SDS at 65°C for 30 min. Data were processed using a Fujix 2000 Bio-image analyzer.
Preparation of Extracts from Cells and Tissues-For immunodetection of MTH1 protein, extracts were prepared as follows. CCE cells and tissue samples from mice perfused with phosphate-buffered saline were homogenized at 4°C in 5 volumes of radioimmune precipitation assay buffer containing 0.5 mM phenylmethylsulfonyl fluoride and 0.5 g/ml each of leupeptin, pepstatin, and chymostatin, followed by sonication on ice to complete disruption of the cells. The lysates were directly applied to both immunoprecipitation and slot blot analysis to determine DNA contents.
Immunodetection of Mouse MTH1 Protein-Immunoprecipitation was done as described by Harlow and Lane (30). Briefly, 1 ml of precleared extract was incubated at 4°C overnight with an excess amount of antibody anti-MTH1 (5 g) or normal rabbit immunoglobulin G (10 g), and protein A-Sepharose CL-4B was used to collect the immunocomplex. After washing five times with 1 ml of radioimmune precipitation assay buffer, the precipitated immunocomplex or antigens in cell extracts were separated on 15% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter, and analyzed by Western blotting using anti-MTH1 (2.5 g/ml). As the control, 1 ng of purified mouse MTH1 protein was used.
Determination of the DNA Content-The DNA contents of CCE cells and various tissue samples were determined by slot blot hybridization using a 1.1-kilobase SacI/BamHI fragment of plasmid pA1 that contained mouse repetitive IAP elements (31). One hundred l of those extracts or genomic DNA of CCE cells were rotated with 10 l of 4 M NaOH (denaturation solution) at room temperature for 15 min. After adding 110 l of 2 M ammonium acetate (neutralization solution), the mixture was centrifuged at 12,000 ϫ g for 10 min. The supernatant was slot blotted onto a nitrocellulose membrane (BA-85, Schleicher & Schuell), using BIO-DOT, SF. Prehybridization and hybridization were carried out at 65°C in a preparation containing 6 ϫ SSC, 5 ϫ Denhardt's solution, 0.5% SDS, 1 mM EDTA, and 100 g/ml heat-denatured salmon sperm DNA. The filter was washed for 1 h in 0.1 ϫ SSC and 0.5% SDS at 65°C. The data were processed using a Fujix 2000 Bioimage analyzer.
Other Methods-[␣-32 P]8-Oxo-dGTP was prepared as previously described (21). Protein concentration was determined using the Bio-Rad protein assay with BSA as a standard (32).

RESULTS
cDNA for Mouse 8-Oxo-dGTPase-A mouse cDNA library, derived from an embryonic stem cell line, was screened for the mouse 8-oxo-dGTPase cDNA sequence, using as a probe a 560-bp NcoI/BamHI DNA fragment which contains the coding region for human MTH1 protein. Twelve out of 5 ϫ 10 5 clones screened showed a strong, positive hybridization signal. After examining sizes of the inserted DNA fragments, four clones were chosen, and their inserts were subcloned into plasmid pBluescript II KS ϩ . Clone M-2 contains the 5Ј-most region of the cDNA, while the other three, M-1, M-8, and M-12, carry the 3Ј-furthermost region (Fig. 1).
Sequences of both strands were determined for more than 90% of the cDNA region (Fig. 2). Included within the approximately 950-bp cDNA is an open reading frame of 471 bp, defined by an ATG initiation codon (nucleotides 99 -101) and a TAA stop codon (nucleotides 567-569), corresponding to a protein composed of 156 amino acid residues. In the 3Ј-untranslated region of 380 nucleotides, a putative polyadenylation signal sequence, AATAAA, was found 14 nucleotides upstream from the poly(A) stretch.
The amino acid sequence deduced from the nucleotide sequence is also shown in Fig. 2. The sequence for the mouse 8-oxo-dGTPase shows 83% identity with that determined for the human enzyme, while the nucleotide sequence for the coding region has 81% identity with that for the human counterpart. Among the total 156 amino acid residues, 27 are replaced by different amino acid residues in the human protein (see Fig. 7).
Expression of Mouse cDNA in E. coli Cells-To confirm that the mouse cDNA we isolated indeed encodes 8-oxo-dGTPase, the cDNA was expressed in E. coli MK602 (mutT Ϫ ) cells, which are devoid of their own 8-oxo-dGTPase activity. To achieve a high level of expression, the cDNA was placed downstream of the strong trc promoter in pTrc99A, and the transformed cells were incubated in the presence of IPTG. When a cell-free extract of cells carrying pTK1 with the cDNA was applied to 15% SDS-polyacrylamide gel electrophoresis, we noted a distinct band corresponding to the 18-kDa protein (Fig. 3A, lane 2),, and here the molecular mass was close to the 17,896 Da of the protein calculated from the predicted amino acid sequence of the cDNA. The band cross-reacted with an anti-human MTH1 polyclonal antibody, anti-MTH1, on Western blot analysis (Fig.   3B, lane 2). Furthermore, a significantly high level of 8-oxo-dGTPase activity was found in the extract when the activity was measured by hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP (Fig. 3C).
The level of expression for the mouse cDNA was considerably higher than that for the human cDNA. When the human cDNA was expressed under the same conditions, no distinct band corresponding to the position of the human enzyme was detected on SDS-polyacrylamide gel electrophoresis analysis (Fig.  3A, lane 3). When the extract of cells carrying the human cDNA was applied at 50 times over that of cells carrying the mouse cDNA, a band for the human enzyme was detected on Western blot analysis using anti-MTH1 (Fig. 3B, lane 3). Thus, approximately a 40-fold larger amount of the mouse enzyme was produced in E. coli cells, as compared with the human enzyme. For this estimation, we used partially purified human and mouse MTH1 TrpE fusion protein to assess the recognition sensitivity of anti-MTH1 antibody against human and mouse MTH1 proteins, respectively (data not shown).
Suppression of the mutT Mutator by Expression of the Mouse cDNA-We examined the effects of cDNA expression on the level of spontaneous mutation frequency toward rifampicin resistance in mutT Ϫ cells. As shown in Table I, the frequency of mutation of E. coli MK602 (mutT Ϫ ) cells carrying the cDNA was as low as that of wild type cells. Such being the case, the mouse MTH1 protein probably functions in E. coli cells to prevent the occurrence of spontaneous mutations caused by accumulation of 8-oxo-dGTP in the nucleotide pool.
Northern Blot Analysis-The size of mouse MTH1 mRNA was estimated by Northern blot hybridization. The poly(A) ϩ RNAs from mouse fibroblast cell line, NIH3T3, and the mouse embryonic stem cell line, CCE, were hybridized with a randomprimed mouse cDNA probe. One major band of about 1.2 kilo- bases was detected (Fig. 4A), and this size of the transcript corresponds to that estimated from the cDNA sequence. However, since the size of cDNA is slightly smaller than that of the mRNA detected by Northern blot analysis, it is likely that the cDNA lacks a certain region of the poly(A) stretch. The 1.2kilobase band was detected in various mouse tissue samples, including kidney, liver, and small intestine (Fig. 4B).
MTH1 Protein in Mouse Tissues-On incubation with extracts of mouse tissues in the presence of Mg 2ϩ , 8-oxo-dGTP was rapidly degraded to 8-oxo-dGMP. However, as there was concomitant formation of 8-oxo-dGDP in the reaction, the extracts probably contained nonspecific nucleoside triphosphatase activity (21). Western blot analysis was the method of quantifying MTH1 protein in tissue samples, and only a faint band for the protein was detected over nonspecific interfering bands. Thus, we made use of immunoprecipitation prior to Western blotting. To optimize the experimental conditions, we analyzed an extract of CCE, a mouse embryonic stem cell line, which contains a relatively large amount of 8-oxo-dGTPase protein. As shown in Fig. 5, a distinct band for MTH1 protein was detected on the blots, and the intensities of signals, quantified by an image analyzer, were proportional to amounts of the extract applied.
Tissue samples were obtained from mice perfused with phosphate-buffered saline, and extracts were prepared in the presence of protease inhibitors. Extracts containing 3 mg of protein were incubated with anti-human MTH1 antibody, anti-MTH1 (5 g), and the immunocomplexes were collected and subjected to Western blot analysis using anti-MTH1. As shown in Fig. 6,   FIG. 3. Expression of mouse cDNA in E. coli cells. E. coli strain MK602 (mutT Ϫ ) was transformed with plasmid pTK1 carrying mouse 8-oxo-dGTPase cDNA or vector plasmid, pTrc99A. Cells with plasmid p24T carrying human 8-oxo-dGTPase cDNA was also used for comparison of expression levels of mouse and human 8-oxo-dGTPase cDNA in E. coli. The cells were grown to A 550 ϭ 0.6 -0.8, at which 1 mM IPTG was added. Aliquots of the cultures were taken at 6 h after IPTG induction, and cell-free extracts were prepared for gel electrophoresis analysis, Western blot analysis, and assay of the enzyme activity.   high intensity bands were detected for samples derived from mouse thymus, liver, spleen, kidney, testis, and large intestine. Less but significant amounts of MTH1 protein are present in brain, heart, lung, and stomach. Although there was no detectable band for the small intestine on Western blot analysis, a band in Northern blot analysis was clearly detected in the sample derived from this organ (see Fig. 4B); MTH1 protein might be cleaved by proteases.
Intensities of the bands were quantified using an image analyzer, and numbers of MTH1 protein molecules per 1 mg of total tissue protein were estimated. Using slot blot hybridization analysis, DNA contents of these tissue samples were also determined, and values per 1 g of DNA were calculated and are summarized in Table II. When comparing amounts of MTH1 based on protein content, thymus, liver, and large intestine had the highest levels. On the basis of cellular DNA content, the highest value was obtained for the liver. Content of MTH1 protein in CCE cells was exceedingly high by comparison. A single cell of mouse embryonic stem line apparently contains 1.65 ϫ 10 5 molecules of MTH1 protein. DISCUSSION 8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxo-dGDP (33). Human cells contain a powerful nucleoside diphosphate kinase activity that converts ribo-and deoxyribonucleoside diphosphates, including 8-oxo-dGDP, to the corresponding nucleoside triphosphates (33). Thus, 8-oxo-dGDP, formed either by oxidation of dGDP or by reduction of 8-oxo-GDP, can be converted 8-oxo-dGTP. Once 8-oxo-dGTP is formed, it can be incorporated into cellular DNA to yield transversion mutation. 8-Oxo-dGTPase is present in bacteria and mammalian cells and appears to function so as to prevent this misincorporation. The enzyme specifically hydrolyzes 8-oxo-dGTP to the monophosphate, and the 8-oxo-dGMP thus formed cannot be rephosphorylated. Guanylate kinase that acts on both GMP and dGMP for phosphorylation is totally inactive for 8-oxo-dGMP (33). By the action of nucleotidase, 8-oxo-dGMP is further degraded to 8-oxo-deoxyguanosine, a form readily excretable through the cellular membrane.
Biological significance of 8-oxo-dGTPase has been established based on the use of E. coli mutants defective in the mutT gene (18 -20). Lack of the gene causes an increased level of A:T to C:G transversion thousands of fold over the wild type cells.  In the present study, we obtained evidence that the level of spontaneous mutation frequency of mutT Ϫ cells, which was elevated, reverted to normal when cDNA for mouse 8-oxo-dGTPase was expressed in such cells. It seems likely that mouse 8-oxo-dGTPase has the same antimutagenic capacity as the E. coli MutT protein.
When the human cDNA was expressed in E. coli mutT Ϫ cells, only a partial suppression of the mutator phenotype was achieved, 8-fold reduction in spontaneous mutation frequency from the level of mutT Ϫ cells but 50 times higher level as compared with mutT ϩ cells (22). In the case of expression of the mouse cDNA, an almost complete suppression occurred, as shown in Table I. This difference may be due to the amounts of the two types of mammalian enzymes produced in E. coli cells. Fortyfold larger amounts of the mouse enzyme were produced in E. coli cells as compared with the case of the human enzyme, although essentially the same vector/expression system was used. It is likely that the mouse enzyme has structural features that are more readily formed and retained in bacterial cells.
It should be noted here that, for the complete suppression of mutT mutation, an exceedingly large amount of the mouse enzyme is needed compared with the authentic MutT protein.
One possible explanation for this inefficiency may be differences in intrinsic catalytic properties of the bacterial and mammalian enzymes. The K m value for the E. coli enzyme is 0.48 M (18), while that for the mammalian enzyme is 12.5 M (21). Thus, the E. coli enzyme more efficiently degrades 8-oxo-dGTP than do the mammalian enzymes.
Another aspect to be considered is the intracellular localization of the enzyme. Enzymes involved in nucleotide metabolism are located mostly in a region where DNA synthesis occurs (34). Hence, the authentic E. coli enzyme may have specific structural features to facilitate proper subcellular localization. Foreign enzymes are likely to be devoid of such signals and may not be able to function as well as the native enzymes.
The 8-oxo-dGTPase protein, originally isolated from E. coli, is composed of 129 amino acid residues (35). Recently, analogous genes were found in two distantly related bacteria, Proteus vulgaris and Streptococcus pneumoniae (36,37). The products of the latter two genes carry enzyme activity specifically degrading dGTP to dGMP and are structurally related to the E. coli MutT protein. Alignment of the sequences of these five mammalian and bacterial enzymes revealed that all carry a highly similar sequence in almost the same region (Fig. 7). In the conserved region (from the 36th to the 58th amino acid for the mouse enzyme), 10 among 23 amino acid residues are identical. Thus, it is reasonable to assume that this region constitutes an active center for the enzyme. The secondary structure of the E. coli MutT protein, as elucidated by NMR analysis (38), supports this view.
When examining the distribution of 8-oxo-dGTPase protein in mice, all organs except the small intestine contained substantial amounts of the protein on Western blot analysis, the highest value was seen in the liver when comparisons were made on the basis of either protein or DNA content. Even a higher level of the enzyme activity was detected in embryonic stem cell line CCE, with an intense proliferative capacity. High oxygen consumption may correlate with high levels of oxidative damage (39), and the level of expression of the MTH1 gene might be regulated, in this context.
To better understand the roles of mammalian 8-oxo-dGT-Pase in the control of spontaneous mutagenesis as well as carcinogenesis, mice defective in their own MTH1 gene are needed. Cloning of cDNA is a first step toward this goal followed by characterization of the genomic sequence. The 8-oxo-dGTPase enzyme is part of an elaborate system of defenses of organisms against oxidative damage to genetic materials. It is of interest to determine when and in which organs of the defective mice such abnormalities could occur. Related studies are in progress in our laboratory. Gaps were inserted to maximize homology. Amino acids residues which are the same as those for the mouse enzyme are boxed, and those conserved through these five species are shadowed.