MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis

Maki, Hisaji; Sekiguchi, Mutsuo

doi:10.1038/355273a0

Cited by 865 publications

(709 citation statements)

References 15 publications

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“…coli cells contain three MutT-type proteins (MutT, Orf135, and Orf17) that catalyze the hydrolysis of 8-OH-dGTP in vitro [13][14][15]. When 8-OH-dGTP was introduced into E. coli strains lacking MutT or Orf135, and a strain expressing the antisense RNA for Orf17, a phenotype was observed only for mutT cells [18,19,42].…”

Section: In Addition Rai Et Al Recently Reported That the Suppressimentioning

confidence: 99%

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

Hori

Satou

Harashima

et al. 2010

Free Radical Biology and Medicine

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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. 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...

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Section: In Addition Rai Et Al Recently Reported That the Suppressimentioning

confidence: 99%

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

Hori

Satou

Harashima

et al. 2010

Free Radical Biology and Medicine

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“…DNA polymerases incorporate 8-OH-dGTP opposite C and A in vitro [7,[16][17][18][19][20][21][22][23]. In terms of the accumulation of 8-OH-Gua in DNA, the contribution of 8-OH-dGTP from the nucleotide pool and the direct oxidation of G bases in DNA has been reported to be almost equal [24].…”

Section: Introductionmentioning

confidence: 99%

“…Nunoshiba et al reported that the mutations found in an Escherichia coli strain lacking superoxide dismutases and a repressor for iron-uptake systems were A:TC:G and G:CT:A transversions, and concluded that the former was caused by 8-OH-dGTP, based on a variety of experimental data [25]. The E. coli MutT protein catalyzes the hydrolysis of 8-OH-dGTP [16], and A:TC:G transversions are induced with high frequency in mutT mutant strains [24,26,27], indicating the importance of 8-OH-dGTP as a source of mutations. Moreover, the presence of mammalian homologues of MutT (MTH1 proteins) supports this speculation [28].…”

Section: Introductionmentioning

confidence: 99%

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

Satou

Kawai

Kasai

et al. 2007

Free Radical Biology and Medicine

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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 co-introduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells. 8-OH-dGTP induced

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“…The damaged DNA precursors formed in the nucleotide pool would be hydrolyzed to the corresponding monophosphates by nucleotide pool sanitization enzymes before they are incorporated into DNA. E. coli MutT and its functional homologues in yeast Saccharomyces cerevisiae (YLR151c) and mammalian cells (MTH1) hydrolyze 8-OH-dGTP [10][11][12]. Nucleotide pool sanitization activities have been identified for two E. coli proteins, Orf135 and Orf17 [13,14].…”

Section: Introductionmentioning

confidence: 99%

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

et al. 2007

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Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2´-deoxyguanosine 5´-triphosphate and 2-hydroxy-2´-deoxyadenosine 5´-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H 2 O 2 , and the rpoB mutant frequencies were calculated. The frequency of the H 2 O 2 -induced mutations was increased in all of the strains tested; however, the increase was threeto four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.

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MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis

Cited by 865 publications

References 15 publications

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

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

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

UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides

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