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

Abstract

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. 3Normal 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][2][3][4][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 1-10% 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][10][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] Mutagenesis experiments293T cells (3 X 10 4 cells) were plated into 24-well dishes and were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, at 37°C under a 5% CO 2 atmosphere for 24 hr. siRNAs (7.2 pmol each) were mixed with Lipofectamine (Invitrogen) and introduced into the cultured 293T cells according to the supplier's recommendations. In the triple knock-down experiment, an siRNA cocktail (MTH1; 3.6 pmol, MTH2; 7.2 pmol and NUDT5; 3.6 pmol) was used.After 24 Quantitative RT-PCR analysis of mRNATotal RNA was extracted from 293T cells using an RNeasy Mini Kit (Qiagen) combined with RNase-free DNase I (Takara, Otsu, Japan), for the degradation of the genomic DNA in the total RNA samples. First-strand cDNA synthesis was performed with 500 ng of t...

Mutations induced by 8-hydroxyguanine (8-oxo-7,8-dihydroguanine), a representative oxidized base, in mammalian cells

“…Both the supF plasmid DNA and dG O TP were introduced into cells in which the expression of each protein was knocked-down. The A:T➔C:G substitution mutations induced by dG O TP were higher in the knockdown cells than in control cells [54]. The increase in the induced mutation was more evident in the triple knockdown cells.…”

Oxidation in the nucleotide pool, the DNA damage response and cellular senescence: Defective bricks build a defective house

“…Thus, in tumor cells, reducing the capacity to eliminate oxidized deoxyribonucleotides is likely to prove a novel and promising chemotherapeutic strategy, particularly in combination with drugs that induce oxidative stress. In a similar vein, assessing the levels and activity of MTH2 [160] and NUDT5, an enzyme that hydrolyzes 8-oxo-dGDP and 8-hydroxy-dADP [161], in cancer cells and determining their roles in inhibiting oxidative stress-induced DDR can provide additional therapeutic targets in the nucleotide pool sanitization pathways.…”

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1

“…MTH1-a member of the family of hydrolases that eliminate oxidized precursors from the dNTP pool (Ishibashi et al, 2003)-degrades both 8-oxodGTP and 8-oxoGTP and prevents 8-oxoG accumulation in DNA and RNA (Sakumi et al, 1993;Hayakawa et al, 1999). MTH1 inactivation is associated with a mutator phenotype in mouse cells (Hori et al, 2010) and increased cancer incidence in mice (Tsuzuki et al, 2001). Recent findings link MTH1 deficiency to neurodegeneration.…”