(Received for publication, May 8, 1995; and in revised form, August 16, 1995)
From the
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), ()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, 9, 10, 11, 12) . MutM protein removes 8-oxoguanine paired with cytosine 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, 19, 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 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.
A pair
of isogenic E. coli strains, MK601 (mutT) and MK602 (mutT
), derived from AB1157 (12) were transformed with pTrc99A or pTK1 and grown overnight
at 30 °C in LB medium containing 50 µg/ml ampicillin. The
cultures were diluted with fresh medium and grown at 37 °C until A
= 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
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.
Figure 1: Organization of cDNA for mouse 8-oxo-dGTPase and strategy for DNA sequencing. The upper panel shows organization of the cDNA with appropriate restriction sites. Closed and open boxes indicate the coding region of cDNA and flanking 5`- and 3`-untranslated regions, respectively. The regions carried by clones M-2, M-8, M-1, and M-12 are shown by shadowed bars. The lower panel shows the strategy used for DNA sequencing. Horizontal arrows show directions and regions of the sequence analyzed.
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 3`-untranslated region of 380 nucleotides, a putative polyadenylation signal sequence, AATAAA, was found 14 nucleotides upstream from the poly(A) stretch.
Figure 2: Nucleotide sequence of cDNA for mouse 8-oxo-dGTPase and its deduced amino acid sequence. Nucleotides and deduced amino acid residues are numbered on the left. The amino acid sequence is numbered from the presumed initiator methionine. The termination codon is marked by dots and a putative polyadenylation signal, AATAAA, by a thick underline.
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).
Figure 7: Comparison of the predicted amino acid sequences of MTH1-related proteins from various organisms. The deduced amino acid sequences of MTH1 related proteins from human (H), mouse (M), S. pneumoniae (S), E. coli (E), and P. vulgaris (P) are shown. 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.
Figure 3:
Expression of mouse cDNA in E. coli cells. E. coli strain MK602 (mutT) 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
= 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. A,
Coomassie Brilliant Blue staining of SDS-polyacrylamide gels after
electrophoresis. Positions and sizes in kilodaltons of marker proteins
are shown on the left side of the panel. Lane 1, 70
µg of protein of an extract of cells carrying pTrc99A (vector); lane 2, 70 µg of protein of an extract of cells carrying
pTK1 (mouse cDNA); lane 3, 70 µg of protein of an extract
of cells carrying p24T (human cDNA). The arrow indicates the
position of the mouse MTH1 protein. B, Western blot analysis
using the anti-human MTH1 antibody, anti-MTH1. Positions and sizes in
kilodaltons of marker proteins are shown on the left side of
the panel. Lane 1, 5 µg of protein of an extract of cells
carrying pTrc99A; lane 2, 0.1 µg of protein of an extract
of cells carrying pTK1 and 4.9 µg of protein of an extract of cells
carrying pTrc99A; lane 3, 5 µg of protein of an extract of
cells carrying p24T. Note that a 50 times larger amount of extract was
used for analysis of the human enzyme as compared with that for the
mouse enzyme. C, 8-Oxo-dGTPase activity in cell-free extracts.
One unit of activity was defined as the amount of enzyme that produced
1 pmol of 8-oxo-dGMP per min at 30 °C.
, an extract of cells
transformed with pTK1 (mouse cDNA);
, an extract of cells
transformed with pTrc99A (vector).
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).
Figure 4:
Northern blot analysis of the mouse MTH1 gene product. A, Northern blot analysis of
poly(A) RNAs from mouse cell lines. Total RNAs were
extracted from mouse cell lines, NIN3T3 and CCE, and 3 µg each of
poly(A)
RNAs were subjected to blotting analysis by
using a 503-bp fragment from pTK1, carrying the coding sequence for
mouse MTH1 cDNA as a probe. Lane 1, NIH3T3 cell; lane
2, CCE cell. B, Northern analysis of total RNAs (20
µg) from various mouse tissues. Lane 1, kidney; lane
2, liver; lane 3, small intestine. To quantify the
amounts of RNAs, the blot was reprobed with 18 S ribosomal RNA gene (lower panel). The position of RNA size markers are shown on
the left side of the panel.
Figure 5:
Quantitative immunoprecipitation of mouse
MTH1 protein. A, quantitative immunoprecipitation of mouse
MTH1 protein. Various amounts of precleared extract from mouse CCE
cells were reacted with anti-MTH1 (5 µg) overnight. The sample used
here was derived from 1 10
cells and contained
roughly 3 mg of protein. Molecular masses of marker proteins are shown
on the left side of the panel. Lane 1,
immunoprecipitant of the extract derived from 2
10
cells (60 µg); lane 2, immunoprecipitant of the
extract from 5
10
cells (150 µg); lane
3, immunoprecipitant of the extract from 1
10
cells (300 µg). B, lineality of Western blotting
signals. The intensities of the bands were quantified by a Fujix 2000
Bio-image analyzer and the value for lane 3 of Panel A was defined as 100. The blotting experiments were done three
times.
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 µg), and the immunocomplexes were collected and subjected to Western blot analysis using anti-MTH1. As shown in Fig. 6, 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.
Figure 6: Contents of MTH1 protein in various mouse tissues. Precleared lysates (3 mg of protein) from various mouse tissues were incubated with anti-MTH1 (5 µg), and the immunocomplexes were subjected to Western blot analysis using anti-MTH1 (2.5 µg/ml). As controls, precleared extracts from CCE cells (300 µg) were reacted with normal rabbit IgG (10 µg) or anti-MTH1 (5 µg). Molecular masses of marker proteins are shown on the left side of the panel. Lane 1, a purified mouse MTH1 protein; lane 2, CCE (IgG); lane 3, CCE (anti-MTH1); lane 4, brain; lane 5, thymus; lane 6, heart; lane 7, lung; lane 8, liver; lane 9, spleen; lane 10, kidney; lane 11, testis; lane 12, large intestine; lane 13, small intestine; lane 14, stomach.
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 µg of
DNA were calculated and are summarized in Table 2. 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 10
molecules of MTH1 protein.
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, 19, 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 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 cells, only a
partial suppression of the mutator phenotype was achieved, 8-fold
reduction in spontaneous mutation frequency from the level of mutT
cells but 50 times higher level as
compared with mutT
cells(22) . In the
case of expression of the mouse cDNA, an almost complete suppression
occurred, as shown in Table 1. 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 value for the E. coli enzyme is 0.48
µM(18) , while that for the mammalian enzyme is
12.5 µM(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-dGTPase 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D49956 [GenBank]