Suppression of mutagenesis by 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate) by human MTH1, MTH2, and NUDT5

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To assess the functions of the three human MutT-type enzymes, MTH1, MTH2, and NUDT5, mutation induction by an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate), was examined using human 293T cells treated with their specific siRNAs. 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. Escherichia coli cells were transformed with the DNAs replicated in the treated cells. The knock-downs of the MTH1, MTH2, and NUDT5 proteins increased the A:T→C:G substitution mutations induced by 8-OH-dGTP. In addition, the increase in the induced mutation frequency was more evident in the triple knock-down cells. These results indicate that all three of the human MTH1, MTH2, and NUDT5 proteins act as a defense against the mutagenesis induced by oxidized dGTP.Normal cellular metabolism produces endogenous reactive oxygen species (ROS). ROS are generated as byproducts of the mitochondrial electron transport chain, and certain cellular enzymes also generate ROS. Moreover, ROS are produced by environmental mutagens/carcinogens, including ionizing radiation and ultraviolet light. The formation of ROS leads to the oxidation of cellular components and disturbs their normal functions. The formation of oxidized DNA lesions is one of the causative factors of mutagenesis, carcinogenesis, neurodegeneration, and aging [1-5]. DNA precursors (2'-deoxyribonucleotides) are also subjected to oxidative damage. The formation of oxidized DNA precursors is a potential source of mutagenesis [6]. 8-Hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate) is the major oxidation product of dGTP in in vitro oxidation reactions [7]. 8-OH-dGTP was reportedly present at a concentration of 1｡V10% relative to the unmodified dGTP in the mitochondrial nucleotide pool [8]. This oxidized form of dGTP is highly mutagenic in living cells when added exogenously [9-11] and is expected to act as an endogenous mutagen. Nucleotide pool sanitization is an important means by which organisms prevent the mutagenesis caused by damaged DNA precursors [6,12]. The Escherichia coli MutT (NudA) protein, encoded by the mutator gene mutT, was the first enzyme found to degrade an oxidized DNA precursor (8-OH-dGTP) in vitro [13]. Moreover, at least two E. coli MutT-type proteins, Orf135 (NudG) and Orf17 (NudB), in addition to MutT, catalyze the hydrolysis of oxidized DNA precursors in vitro [14,15]. E. coli strains lacking MutT and Orf135 exhibit a mutator phenotype [16-19], indicating the importance of nucleotide pool sanitization to prevent mutagenesis by oxidized DNA precursors. Mammalian cells also possess MutT-type enzymes. The MTH1 (NUDT1) protein catalyzes the hydrolysis of various oxidized DNA precursors, including 8-OH-dGTP, and greater numbers of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice than wild-type mice [20,21]. MTH2 (NUDT15) degrades 8-OH-dGTP in vitro, and the expression of the cDNA encoding MTH2 significantly reduced the elevated spontaneous mutation frequency in E. coli mutT cells [22]. The NUDT5 protein is unique, because the hydrolysis of 8-hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-diphosphate) by this protein is much more efficient than that of 8-OH-dGTP [23,24]. However, the expression of NUDT5 in mutT-deficient E. coli mutant cells decreases the spontaneous mutation frequency to the normal level, suggesting the importance of its 8-OH-dGDPase activity in nucleotide pool sanitization [23]. Thus, it is quite important to examine whether the three proteins prevent the mutations induced by 8-OH-dGTP in mammalian (human) cells.Recently, we showed that 8-OH-dGTP specifically induced A:T→C:G transversions in live simian and human cells [10,11]. In this study, to assess the contributions of these MutT-type proteins to the prevention of the mutagenesis caused by 8-OH-dGTP in human cells, we suppressed their expression by siRNAs and introduced 8-OH-dGTP into the knocked-down cells. The knock-downs of all of the MutT-type proteins enhanced the A:T→C:G substitution mutations, suggesting their roles as functional nucleotide pool sanitization enzymes.
2010-05-01

Suppression of mutagenesis by 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate) by human MTH1, MTH2, and NUDT5

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