Daisuke Tsuchimoto

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids.

Abstract 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.

Oxidative damage in nucleic acids and Parkinson's disease

Oxidative DNA lesions, such as 8‐oxoguanine (8‐oxoG), accumulate in nuclear and mitochondrial genomes during aging, and such accumulation can increase dramatically in patients with Parkinsons disease (PD). To counteract oxidative damage to nucleic acids, human and rodents are equipped with three distinct enzymes. One of these, MTH1, hydrolyzes oxidized purine nucleoside triphosphates, such as 8‐oxo‐2′‐deoxyguanosine triphosphate and 2‐hydroxy‐2′‐deoxyadenosine triphosphate, to their monophosphate forms. The other two enzymes are 8‐oxoG DNA glycosylase encoded by the OGG1 gene and adenine/2‐hydroxyadenine DNA glycosylase encoded by the MUTYH gene. We have shown a significant increase in 8‐oxoG in mitochondrial DNA as well as an elevated expression of MTH1, OGG1, and MUTYH in nigrostriatal dopaminergic neurons of PD patients, suggesting that the buildup of these lesions may cause dopamine neuron loss. We established MTH1‐null mice and found that MTH1‐null fibroblasts were highly susceptible to cell death caused by H2O2 characterized by pyknosis and electron‐dense deposits in the mitochondria, and that this was accompanied by an ongoing accumulation of 8‐oxoG in nuclear and mitochondrial DNA. We also showed that MTH1‐null mice exhibited an increased accumulation of 8‐oxoG in striatal mitochondrial DNA, followed by more extreme neuronal dysfunction after 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine administration than that of wild‐type mice. In conclusion, oxidative damage in nucleic acids is likely to be a major risk factor for Parkinsons disease, indicating that a solid understanding of the defense mechanisms involved will enable us to develop new strategies for protecting the brain against oxidative stress.

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs.

Oxidative base lesions, such as 8‐oxoguanine (8‐oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8‐oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8‐oxoG DNA glycosylase in OGG1‐null mouse cells. The accumulation of 8‐oxoG in nuclear DNA caused poly‐ADP‐ribose polymerase (PARP)‐dependent nuclear translocation of apoptosis‐inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single‐strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8‐oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.

The defense mechanisms in mammalian cells against oxidative damage in nucleic acids and their involvement in the suppression of mutagenesis and cell death

To counteract oxidative damage in nucleic acids, mammalian cells are equipped with several defense mechanisms. We herein review that MTH1, MUTYH and OGG1 play important roles in mammalian cells avoiding an accumulation of oxidative DNA damage, both in the nuclear and mitochondrial genomes, thereby suppressing carcinogenesis and cell death. MTH1 efficiently hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP and 2-hydroxy (OH)-dATP, to the monophosphates, thus avoiding the incorporation of such oxidized nucleotides into the nuclear and mitochondrial genomes. OGG1 excises 8-oxoG in DNA as a DNA glycosylase and thus minimizes the accumulation of 8-oxoG in the cellular genomes. MUTYH excises adenine opposite 8-oxoG, and thus suppresses 8-oxoG-induced mutagenesis. MUTYH also possesses a 2-OH-A DNA glycosylase activity for excising 2-OH-A incorporated into the cellular genomes. Increased susceptibilities to spontaneous carcinogenesis of the liver, lung or intestine were observed in MTH1-, OGG1- and MUTYH-null mice, respectively. The increased occurrence of lung tumors in OGG1-null mice was abolished by the concomitant disruption of the Mth1 gene, indicating that an increased accumulation of 8-oxoG and/or 2-OH-A might cause cell death. Furthermore, these defense mechanisms also likely play an important role in neuroprotection.

Mutator Phenotype of MUTYH-null Mouse Embryonic Stem Cells

To evaluate the antimutagenic role of a mammalian mutY homolog, namely the Mutyh gene, which encodes adenine DNA glycosylase excising adenine misincorporated opposite 8-oxoguanine in the template DNA, we generated MUTYH-null mouse embryonic stem (ES) cells. In the MUTYH-null cells carrying no adenine DNA glycosylase activity, the spontaneous mutation rate increased 2-fold in comparison with wild type cells. The expression of wild type mMUTYH or mutant mMUTYH protein with amino acid substitutions at the proliferating cell nuclear antigen binding motif restored the increased spontaneous mutation rates of the MUTYH-null ES cells to the wild type level. The expression of a mutant mMUTYH protein with an amino acid substitution (G365D) that corresponds to a germ-line mutation (G382D) found in patients with multiple colorectal adenomas could not suppress the elevated spontaneous mutation rate of the MUTYH-null ES cells. Although the recombinant mMUTYH(G365D) purified from Escherichia coli cells had a substantial level of adenine DNA glycosylase activity as did wild type MUTYH, no adenine DNA glycosylase activity was detected in the MUTYH-null ES cells expressing the mMUTYH(G365D) mutant protein. The germ-line mutation (G382D) of the human MUTYH gene is therefore likely to be responsible for the occurrence of a mutator phenotype in these patients.

Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species: From mitochondria to nuclei

Abstract: In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8‐oxo‐dGTP, 8‐oxo‐dATP, and 2‐hydroxy (OH)‐dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1‐deficient mice and an alteration of MTH1 expression along with the accumulation of 8‐oxo‐dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8‐oxoG DNA glycosylase (OGG1), 2‐OH‐A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous carcinogenesis in OGG1 and MUTYH‐deficient mice. The increased occurrence of lung tumor in OGG1‐deficient mice was completely abolished by the concomitant disruption of the Mth1 gene.

Programmed cell death triggered by nucleotide pool damage and its prevention by MutT homolog-1 (MTH1) with oxidized purine nucleoside triphosphatase.

Accumulation of oxidized bases such as 8-oxoguanine in either nuclear or mitochondrial DNA triggers various cellular dysfunctions including mutagenesis, and programmed cell death or senescence. Recent studies have revealed that oxidized nucleoside triphosphates such as 8-oxo-dGTP in the nucleotide pool are the main source of oxidized bases accumulating in the DNA of cells under oxidative stress. To counteract such deleterious effects of nucleotide pool damage, mammalian cells possess MutT homolog-1 (MTH1) with oxidized purine nucleoside triphosphatase and related enzymes, thus minimizing the accumulation of oxidized bases in cellular DNA. Depletion or increased expression of the MTH1 protein have revealed its significant roles in avoiding programmed cell death or senescence as well as mutagenesis, and accumulating evidences indicate that MTH1 is involved in suppression of degenerative disorders such as neurodegeneration.

ITPase-deficient mice show growth retardation and die before weaning.

Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa−/− mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac α-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa−/− mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.

A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA

2-Hydroxy-2-deoxyadenosine triphosphate (2-OH-dATP), generated by the oxidation of dATP, can be misincorporated by DNA polymerases opposite guanine in template DNA during DNA replication, thus causing spontaneous mutagenesis. We demonstrated that mouse MUTYH (mMUTYH) has a DNA glycosylase activity excising not only adenine opposite 8-oxoguanine (8-oxoG) but also 2-hydroxyadenine (2-OH-A) opposite guanine, using purified recombinant thioredoxin-mMUTYH fusion protein. mMUTYH formed a stable complex with duplex oligonucleotides containing an adenine:8-oxoG pair, but the binding of mMUTYH to oligonucleotides containing a 2-OH-A:guanine pair was barely detectable, thus suggesting that mMUTYH recognizes and interacts with these two substrates in a different manner which may reflect the difference in the base excision repair process for each substrate. Mutant mMUTYH with G365D amino acid substitution, corresponding to a G382D germline mutation of human MUTYH found in familial adenomatous polyposis patients, almost completely retained its DNA glycosylase activity excising adenine opposite 8-oxoG; however, it possessed 1.5% of the wild-type activity excising 2-OH-A opposite guanine. Our results imply that the reduced repair capacity of the mutant hMUTYH(G382D), which inefficiently excises 2-OH-A opposite guanine, results in an increased occurrence of somatic G:C to T:A transversion mutations in the APC gene as well as tumorigenesis in the colon.

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa− mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa− embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa− primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa− MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa− embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa− embryos. However, immortalized Itpa− MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa− MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.