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.3
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
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:TC: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...
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 alpha 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.
To reveal the roles of Y family DNA polymerases in the mutagenesis induced by oxidatively damaged DNA precursors, 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP (8-OH-dGTP) were introduced into Escherichia coli strains deficient in the Y family polymerases, DNA polymerase IV (pol IV, encoded by the dinB gene) and DNA polymerase V (pol V, encoded by the umuDC locus). The mutation induced by 2-OH-dATP, but not that induced by 8-OH-dGTP, occurred less frequently in the dinB- strain than in the wild-type (wt) strain, suggesting the involvement of pol IV in the mutagenesis by 2-OH-dATP. Expression of pol IV from plasmid enhanced the mutagenesis by 2-OH-dATP in the dinB- strain. This enhancement depends on the polymerase activity since the expression of a mutant pol IV lacking the polymerase activity did not increase the mutations induced by 2-OH-dATP. In contrast, both 2-OH-dATP and 8-OH-dGTP caused mutations more efficiently in the umuDC- strain than in the wt strain, suggesting that the umuDC gene products suppressed the mutagenesis by these oxidized DNA precursors. The DNA polymerase activity was not required for the suppressive effects because expression of the umuDC gene products lacking the polymerase activity also suppressed the mutagenesis. These results suggest that the E. coli pol IV was involved in mutagenesis by 2-OH-dATP and that the umuDC gene products play suppressive role(s) in the mutagenesis by damaged nucleotides.
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