ABSTRACT
Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol ß, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.
Subject(s)
Cytotoxins/metabolism , DNA Damage , DNA Polymerase beta/chemistry , DNA Polymerase beta/metabolism , Deoxyguanine Nucleotides/metabolism , Deoxyguanine Nucleotides/toxicity , Mutagenesis , Adenine/chemistry , Adenine/metabolism , Base Pairing , Catalytic Domain , Crystallography, X-Ray , Cytosine/chemistry , Cytosine/metabolism , Cytotoxins/chemistry , Cytotoxins/toxicity , DNA/biosynthesis , DNA/chemistry , DNA Repair , DNA Replication , Deoxyguanine Nucleotides/chemistry , Guanine/analogs & derivatives , Guanine/chemistry , Guanine/metabolism , Humans , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Conformation , Neoplasms/enzymology , Neoplasms/genetics , Oxidation-Reduction , Oxidative Stress , Static Electricity , Substrate Specificity , Time FactorsABSTRACT
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 knockdown 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-knockdown 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.
Subject(s)
DNA Repair Enzymes/metabolism , Mutagenesis/physiology , Phosphoric Monoester Hydrolases/metabolism , Pyrophosphatases/metabolism , 8-Hydroxy-2'-Deoxyguanosine , Deoxyguanine Nucleotides/toxicity , Humans , Mutagens/toxicity , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
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 cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.
Subject(s)
DNA/drug effects , Deoxyguanine Nucleotides/toxicity , Mutagenesis , 8-Hydroxy-2'-Deoxyguanosine , Animals , COS Cells , Cations/chemistry , Chlorocebus aethiops , DNA/genetics , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Escherichia coli Proteins/genetics , Liposomes/chemistry , Mutation , Plasmids/chemistry , Plasmids/drug effects , Plasmids/genetics , TransfectionABSTRACT
The remarkable carcinogenic activity of 6-nitrochrysene (6-NC) in several animal models, and its environmental presence, suggest its potential importance with regard to human cancer development. Depending on the bioassay model, 6-NC can be activated by simple nitro reduction, ring oxidation, or by a combination of ring oxidation and nitro reduction. Only the first pathway has been clearly established. Thus, this study purports to unequivocally define the other pathways. Toward this end, we report for the first time the synthesis of anti-1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-6-nitrochrysene (6-NCDE), a likely ultimate carcinogenic metabolite of 6-NC. Also, we describe our initial investigation of its binding with calf thymus DNA, 2'-deoxyguanosine-5'-monophosphate (2'-dGuo), and 2'-deoxyadenosine-5'-monophosphate (2'-dAdo) in vitro. These adduct markers were then employed for comparison with those obtained in the rat after in vivo treatment with 6-NC. On the basis of the results, it appears that the major adduct formed in the liver of rats treated with 6-NC is not derived from 6-NCDE.
Subject(s)
Carcinogens/chemical synthesis , Chrysenes/chemical synthesis , DNA/metabolism , Deoxyadenine Nucleotides/metabolism , Deoxyguanine Nucleotides/metabolism , Animals , Carcinogens/metabolism , Carcinogens/toxicity , Cattle , Chromatography, High Pressure Liquid , Chrysenes/metabolism , Chrysenes/toxicity , DNA/drug effects , DNA Adducts/biosynthesis , Deoxyadenine Nucleotides/toxicity , Deoxyguanine Nucleotides/toxicity , Magnetic Resonance Imaging , Thymus Gland/chemistry , Thymus Gland/metabolismABSTRACT
The effects of deoxynucloside triphosphates (dNTPs) on the frequency of sister-chromatid exchange (SCE) was studied in human peripheral lymphocytes. Treatment with each dCTP and dTTP did not change the SCE frequency; however, dGTP caused a significant dose-dependent increase in SCE frequency, whereas dATP caused a significantly decreased in SCE frequency. The SCE-increasing effect of the treatment with dGTP could be totally reversed by treatment with equal concentrations of dATP or dCTP. Treatment with a mixture of four equal concentrations of dATP, dGTP, dCTP, dTTP did not alter the SCE frequency. These results suggest that the dNTP pool imbalance was a SCE-effecting factor in human lymphocytes, and that dGTP may be mainly responsible for this effect.
