Specificity of mutations induced by incorporation of oxidized dNTPs into DNA by human DNA polymerase eta

Aberrant oxidation is a property of many tumor cells. Oxidation of DNA precursors, i.e., deoxynucleotide triphosphates (dNTPs), as well as DNA is a major cause of genome instability. Here, we report that human DNA polymerase eta (h Poleta) incorporates oxidized dNTPs, i.e., 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), into DNA in an erroneous and efficient manner, thereby inducing various types of mutations during in vitro gap-filling DNA synthesis. When 2-OH-dATP was present at a concentration equal to those of the four normal dNTPs in the reaction mixture, DNA synthesis by h Poleta enhanced the frequency of G-to-T transversions eight-fold higher than that of the transversions in control where only the normal dNTPs were present. When 8-OH-dGTP was present at an equimolar concentration to the normal dNTPs, it enhanced the frequency of A-to-C transversions 17-fold higher than the control. It also increased the frequency of C-to-A transversions about two-fold. These results suggest that h Poleta incorporates 2-OH-dATP opposite template G and incorporates 8-OH-dGTP opposite template A and slightly opposite template C during DNA synthesis. Besides base substitutions, h Poleta enhanced the frequency of single-base frameshifts and deletions with the size of more than 100 base pairs when 8-OH-dGTP was present in the reaction mixture. Since h Poleta is present in replication foci even without exogenous DNA damage, we suggest that h Poleta may be involved in induction of various types of mutations through the erroneous and efficient incorporation of oxidized dNTPs into DNA in human cells.     

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.     

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.     

Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate)

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.     

2-Hydroxy-dATP is incorporated opposite G by Escherichia coli DNA polymerase III resulting in high mutagenicity

Four kinds of oxidatively damaged DNA precursors, 8-hydroxydeoxyguanosine 5′-triphosphate (8-OH-dGTP), 2-hydroxydeoxyadenosine 5′-triphosphate (2-OH-dATP), 5-hydroxydeoxycytidine 5′-triphosphate (5-OH-dCTP) and 5-formyldeoxyuridine 5′-triphosphate (5-CHO-dUTP), were employed in in vitro gap-filling reactions of the supF gene conducted by the Escherichia coli DNA polymerase III holoenzyme, and these treated DNAs were transfected into various E.coli strains. When the manipulated DNAs were transfected into the repair-proficient strain, supF mutants were obtained much more frequently by the purine nucleotides than by the pyrimidine nucleotides (2-OH-dATP > 8-OH-dGTP >> 5-OH-dCTP ~ 5-CHO-dUTP). This result is in contrast to our previous observation that these four oxidatively damaged nucleotides induce chromosomal gene mutations with similar frequencies when incorporated directly into E.coli. 2-OH-dATP elicited G→T transversions, indicating the formation of G•2-OH-dATP pairs. These results demonstrate that 2-OH-dATP was highly mutagenic in this assay system containing the in vitro DNA synthesis by the E.coli replicative DNA polymerase, in addition to in the in vivo assay system reported previously. Slight increases in the mutant frequencies were observed when alkA (for 8-OH-dGTP and 2-OH-dATP) and mutY (for 2-OH-dATP) strains were used as hosts. This is the first report that clearly shows the formation of G•2-OH-dATP pairs.     

Dual hydrolysis of diphosphate and triphosphate derivatives of oxidized deoxyadenosine by Orf17 (NtpA), a MutT-type enzyme

To determine whether the Orf17 (NtpA) protein of Escherichia coli, a MutT-type enzyme, functions as a hydrolyzing enzyme for a damaged deoxyribonucleotide, we purified the recombinant Orf17 protein and incubated it with oxidized deoxyribonucleotides. Of the deoxyribonucleoside 5'-triphosphates tested, 8-hydroxy-2'-deoxyadenosine 5'-triphosphate was hydrolyzed by this protein. Unexpectedly, the Orf17 protein degraded 8-hydroxy-2'-deoxyadenosine 5'-diphosphate 2.3-fold more efficiently than the corresponding triphosphate. Thus, this protein is the first MutT-type enzyme that hydrolyzes both the triphosphate and diphosphate derivatives of a deoxyribonucleoside, with similar efficiencies. These results suggest that the Orf17 protein may be involved in the hydrolysis of oxidized dATP and dADP.     

Mutagenic effects of 2-hydroxy-dATP on replication in a HeLa extract: induction of substitution and deletion mutations

The mutagenicity of an oxidized form of dATP, 2-hydroxydeoxyadenosine 5′-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP induced mutations in a dose-dependent manner and elicited substitution and deletion mutations. Of the substitutions, a G·C→A·T transition including a tandem (CC→TT) mutation was mainly observed. This result agrees with our previous observation that mammalian DNA polymerase α misincorporates the oxidized nucleotide opposite C, but is in contrast to the finding that 2-OH-dATP elicits G·C→T·A transversions in Escherichia coli. This type of mutation was also elicited, but to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5′-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture, and probably in living cells. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.     

Two DNA polymerases of Escherichia coli display distinct misinsertion specificities for 2-hydroxy-dATP during DNA synthesis

The insertion specificities of an oxidized dATP analogue, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I. In contrast to our previous observation that mammalian DNA polymerase alpha incorporated the oxidized nucleotide opposite T and C, these two E. coli DNA polymerases incorporated 2-OH-dATP opposite T and G on the DNA template. Steady-state kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times more frequently opposite T than opposite G. On the other hand, the incorporation of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2 orders of magnitude more efficient than that opposite G. These results indicate that the misinsertion specificity of 2-OH-dATP differs between replicative and repair-type DNA polymerases, and provide a biochemical basis for the mutations induced by 2-OH-dATP in E. coli.     

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

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

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP), was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T → C:G substitution mutations in the cells. The knock-downs of DNA polymerases η and ζ, and REV1 by siRNAs reduced the A:T → C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast, the knock-down of DNA polymerase ι did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols η plus ζ and REV1 plus DNA pol ζ (but not by that of DNA pol η plus REV1), suggesting that REV1-DNA pol η and DNA pol ζ work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP.     

Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.     

The mutations induced by oxidatively damaged nucleotides, 5-formyl-dUTP and 5-hydroxy-dCTP,in Escherichia coli.

The mutational properties of 5-formyl-2'-deoxyuridine 5'-triphosphate (5-CHO-dUTP) and 5-hydroxy-2'-deoxycytidine 5'-triphosphate (5-OH-dCTP), the major oxidatively damaged pyrimidine nucleotides derived from dTTP and dCTP, respectively, were analyzed by an in vivo assay. 5-CHO-dUTP and 5-OH-dCTP were directly incorporated into Escherichia coli , and their mutagenicities were evaluated by the chromosomal lacI forward mutation assay. The mutation frequencies increased, depending on the dose of these damaged nucleotides, indicating that these nucleotides were incorporated into E.coli and acted as mutagens in vivo . The mutagenicities of 5-CHO-dUTP and 5-OH-dCTP were comparable to that of 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, a major form of dGTP oxidative damage. 5-CHO-dUTP induced G.C to A.T, A.T to G.C and G.C to T.A mutations, and 5-OH-dCTP elicited G.C to A.T, A.T to C.G and G.C to T.A mutations.     

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids

Genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are generated both as byproducts of oxygen respiration or molecular executors in the host defense, and by environmental exposure to ionizing radiation and chemicals. To counteract such oxidative damage in nucleic acids, mammalian cells are equipped with three distinct enzymes. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2'-deoxyguanosine triphosphate and 2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP), to the corresponding monophosphates. We observed increased susceptibility to spontaneous carcinogenesis in MTH1-null mice, which exhibit an increased occurrence of A:T-->C:G and G:C-->T:A transversion mutations. 8-Oxoguanine (8-oxoG) DNA glycosylase, encoded by the OGG1 gene, and adenine DNA glycosylase, encoded by the MUTYH gene, are responsible for the suppression of G:C to T:A transversions caused by the accumulation of 8-oxoG in the genome. Deficiency of these enzymes leads to increased tumorigenesis in the lung and intestinal tract in mice, respectively. MUTYH deficiency may also increase G:C to T:A transversions through the misincorporation of 2-OH-dATP, especially in the intestinal tract, since MUTYH can excise 2-hydroxyadenine opposite guanine in genomic DNA and the repair activity is selectively impaired by a mutation found in patients with autosomal recessive colorectal adenomatous polyposis.     

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.     

NUDT5 hydrolyzes oxidized deoxyribonucleoside diphosphates with broad substrate specificity

The human NUDT5 protein catalyzes the hydrolysis of 8-hydroxy-dGDP. To examine its substrate specificity, four oxidized deoxyribonucleotides (2-hydroxy-dADP, 8-hydroxy-dADP, 5-formyl-dUDP, and 5-hydroxy-dCDP) were incubated with the NUDT5 protein. Interestingly, all of the nucleotides, except for 5-hydroxy-dCDP, were hydrolyzed with various efficiencies. The kinetic parameters indicated that 8-hydroxy-dADP was hydrolyzed as efficiently as 8-hydroxy-dGDP. The hydrolyzing activities for their triphosphate counterparts were quite weak. These results suggest that the NUDT5 protein eliminates various oxidized deoxyribonucleoside diphosphates from the nucleotide pool and prevents their toxic effects.     

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.     

Differential inhibition of DNA polymerases of calf thymus by 9-beta-D-arabinofuranosyladenine-5'-triphosphate.

The effect of 9-beta-D-arabinofuranosyladenine-5'-triphosphate (araATP) on the reactions of DNA polymerases alpha and beta [E.C. 2.7.7.7] purified from calf thymus was examined. The reaction of DNA polymerase alpha was shown to be more sensitive to the inhibition than that of DNA polymerase beta. The K1 value of DNA polymerase beta for araATP was 45 micrometer; 15 times higher than that of DNA polymerase alpha (3 micrometer). The mode of inhibition by araATP was essentially competitive to deoxyadenosine triphosphate (dATP) in the reactions catalyzed by both DNA polymerase alpha and beta using activated DNA as a template-primer. However, in the reactions of the alpha-enzyme, araATP also inhibited the incorporation of deoxyribonucleotides othan than dATP non-competitively.     

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.