Ring-opened 7-methylguanine residues in DNA are a block to in vitro DNA synthesis.

Single-stranded M13mp18 phage DNA was methylated with dimethylsulfate (DMS), and further treated with alkali to ring-open N7-methylguanine residues and yield 2-6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) residues. Nucleotide incorporation during in vitro DNA synthesis on methylated template using E. coli DNA polymerase Klenow fragment (Kf polymerase) was reduced compared to the unmethylated template. Additional treatment of the methylated template with NaOH to generate Fapy residues, further reduced in vitro DNA synthesis compared to the synthesis on methylated templates, which suggested that Fapy residues were a block to in vitro DNA synthesis. Analysis of the termination products on sequencing gels, assuming that synthesis stops one base before a blocking lesion, indicated that arrest of DNA synthesis upon direct alkylation of single-stranded DNA occurred 1 base 3' to template adenine residues in the case of Kf polymerase and 1 base 3' to adenine and cystosine residues for T4 polymerase. When the alkylated templates were treated with NaOH to produce a template which converted all the N7-methylguanine residues to Fapy residues, the blocks to DNA synthesis were still observed one base before adenine residues. In addition to the stops previously observed for the methylated templates, however, new stops occurred one base 3' to template guanine residues for synthesis using both Kf polymerase and T4 polymerase. Fapy residues, therefore, represent a potential lethal lesion which may also arrest in vivo DNA synthesis if not repaired.     

Imidazole open ring 7-methylguanine: an inhibitor of DNA synthesis

Guanine methylated at the N7 position (me7G) is susceptible to cleavage of the imidazole ring yielding: 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (rom7G). DNA synthesis catalysed by E.coli DNA polymerase I, using as templates poly(dGC) containing either me7G or rom7G, show that rom7G blocks DNA chain elongation. It implies a potential killing effect. Furthermore rom7G does not induce mispairing with either dAMP or dTMP. me7G does not affect DNA synthesis. The results suggest that, beside AP-sites, rom7G is a potential killing lesion in cells treated by alkylating agents.     

Human cDNA expressing a functional DNA glycosylase excising 3-methyladenine and 7-methylguanine.

A cDNA expression library from a human cell line was introduced into an E. coli strain deficient in the repair of 3-meAde bases in DNA. E. coli strains deficient in the repair of 3-meAde are unusually sensitive to DNA methylating agents. A plasmid pANPG10 (Alkyl-N-Purine-DNA Glycosylase) was rescued from the library based on its ability to reduce the sensitivity of the mutant strain to methylmethane sulfonate. Crude extracts of the E. coli mutant strain hosting the plasmid pANPG10 release both 3-meAde and 7-meGua from DNA. The longest open reading frame in the sequence codes for a polypeptide of 230 amino acids of molecular weight 25.5 kD, with a pI of 9.1. The derived amino acid sequence of the human 3-meAde-DNA glycosylase has 85% sequence identity with the 3-meAde-DNA glycosylase from rat hepatoma cells.     

A system in mouse liver for the repair of O6-methylguanine lesions in methylated DNA.

An activity from mouse liver with catalyzes the disappearance of O6-methylguanine from DNA methylated with methylnitrosourea has been partially purified by ammonium sulfate fractionation and DNA-cellulose chromatography. The activity does not require divalent metal ions and is not affected by EDTA. It is specific for the repair of O6-methylguanine lesions and does not affect the removal of 7-methylguanine, 7-methyladenine or 3-methyladenine. The disappearance of O6-methylguanine is linear with respect to the concentration of protein and is dependent on incubation temperature. The kinetics and substrate dependence experiments suggest that the protein factor is product-inactivated. Amino acid analysis of hydrolysates of protein obtained after incubation of methylated DNA with the protein factor indicates the presence of radiolabeled S-methyl-L-cysteine, suggesting that during the repair of O6-methylguanine from methylated DNA, the methyl group is transferred to a sulfhydryl of a cysteine residue of a protein. This represents the first such demonstration in a mammalian system.     

Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA.

Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua). The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E. coli DNA polymerase I. The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA. Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base). The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells. Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA. In contrast, mutation frequency at adenine sites yielding A----G transitions was the most frequent event, 60-fold increased over DMS induced mutations. These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria. The results also imply that the Fapy-7-MeGua in E. coli cells is primarily a lethal lesion.     

Depurination from DNA of 7-methylguanine, 7-(2-aminoethyl)-guanine and ring-opened 7-methylguanines

DNA was reacted with dimethyl sulphate and ethyleneimine to afford respective 7-methylguanine and 7-(2-aminoethyl)guanine derivatives. The substituted DNA was boiled in 0.1 M NaCl containing 10 mM phosphate buffer (pH 7.0), and the release of 7-alkylguanines, guanine and adenine was followed. The half-lives of depurination were 1.5 and 4.1 min for 7-(2-aminoethyl)guanine and 7-methylguanine, respectively. 7-Methylguanine was released some 60 times faster than guanine and adenine. When 7-methylguanine-containing DNA was treated in alkali to cause imidazole ring-opening, two products were liberated by boiling the DNA solution. These products were released with apparent half-lives of 69 and 34 min. These ring-opened products isomerized to each other completely within 1 h at 37°C. The isomers had an identical ultraviolet spectrum and they displayed a pK_a of 9.8. When silylated and analysed in gas chromatography-mass spectroscopy the two isomers had an identical molecular weight and fragmentation pattern, consistent with a structural assignment as N⁵-methyl-N⁵-formyl-2,5,6-triamino-4-oxopyrimidine. Only one of the isomers appeared to be present on DNA; the isomerization took place when the ring-opened product was released into solution.     

A viral genome containing an unstable aflatoxin B1-N7-guanine DNA adduct situated at a unique site.

A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability. Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8- (N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B1 (AFB1). A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB1 to form an oligonucleotide containing a single AFB1-N7-Gua (at the underlined guanine). This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI. Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III. The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB1-N7-Gua stability. Characterization procedures indicated that the AFB1-N7-Gua genome was approximately 95% pure with a small (5%) contamination by unmodified genome. This construction scheme should be applicable to other chemically or thermally unstable DNA adducts.     

Crystal and molecular structure of a DNA duplex containing the carcinogenic lesion O6-methylguanine

The crystal and molecular structure of the first DNA duplex containing the carcinogenic lesion O6MeG has been determined to a resolution of 1.9 A and refined to an R factor of 19%. (d[CGC-(O6Me)GCG])2 crystallizes in the left-handed Z DNA form and has crystal parameters and conformational features similar to those of the parent sequence [d(CG)3]2. The methyl groups on O6 of G4 and G10 have C5-C6-O6-O6Me torsion angles of 73 degrees and 56 degrees, respectively, and protrude onto the major groove surface. The base-pairing conformation for the methylated G.C base pairs is of the Watson-Crick type as opposed to a wobble-type conformation that had been proposed in a B DNA fragment. As in other Z DNA structures, a spine of hydration is seen in the minor groove.     

In vivo replication of carcinogen-modified rat liver DNA: increased susceptibility of O6-methylguanine compared to N-7-methylguanine in replicated DNA to S1-nuclease

In order to characterize rat liver DNA replicated $\text{in}\text{vivo}$ on a carcinogen-damaged template, the replicated DNA was treated with S₁-nuclease and the release of (¹⁴C)-dimethyl-nitrosamine induced 0⁶-methylguanine, a lesion associated with miscoding and N-7-methylguanine, a lesion that does not miscode were monitored. The results indicated that both the methylated guanines became susceptible to S₁-nuclease upon replication. However, a greater percentage of 0⁶-methylguanine (22% of the total 0⁶-methylguanine present in the DNA) compared to N-7-methylguanine (4% of the total N-7-methylguanine present in the DNA) was rendered acid soluble by S₁-nuclease. The preferential release of 0⁶-methylguanine compared to N-7-methylguanine from replicated DNA was interpreted to indicate its occurrence in local denatured regions probably generated as a result of misbase pairing.     

Novel synthesis of O6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT)

The human DNA repair protein O⁶-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O⁶-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access for the first time to a wide variety of oligodeoxyribonucleotides (ODNs) containing O⁶-alkylguanines. One such ODN containing O⁶-(4-bromothenyl)guanine is the most potent inactivator described to date with an IC₅₀ of 0.1 nM.     

Steady-state levels of 7-methylguanine increase in nuclear DNA of postmitotic mouse tissues during aging

The major DNA product formed by methylating agents in vitro and in vivo is 7-methylguanine (m7Gua). In untreated rodent genomes, this damage is thought to arise as a consequence of endogenous processes. Using 2 independent HPLC systems and 2 methods of detection, we observed that low levels of m7Gua are present in nuclear DNA of normal 23-month-old postmitotic mouse tissues. We then asked whether the steady-state levels of indigenous m7Gua change as a function of age in these tissues. C57BL/6NNia male mice 11 months, 23 months, and 28 months of age were analyzed. The results showed that in nuclear DNA of brain, liver, and kidney tissues, the steady-state levels of m7Gua increased approximately 2-fold between the young and old age groups. The persistence of N-methyl-N-nitrosourea (MNU)-induced m7Gua in these tissues in treated animals was also studied. Following a 25 mg MNU/kg body weight dose, administered by the intraperitoneal route, m7Gua appeared to be at least partially persistent for a period of up to 20 days. The degree of persistence of m7Gua, however, appeared to be independent of tissue or age. Since m7Gua has intrinsic mutagenic potential and the content of m7Gua is generally a good indicator of overall alkylation damage to DNA, an age-related increase in the steady-state amounts of m7Gua may be relevant to basic mechanisms of aging and carcinogenesis.     

Release of 7-methylguanine residues whose imidazole rings have been opened from damaged DNA by a DNA glycosylase from Escherichia coli.

Double-stranded DNA containing 7-methylguanine residues whose imidazole rings have been opened, i.e. 2,6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine residues, may be prepared by treatment of DNA with dimethyl sulfate followed by prolonged incubation at pH 11.4. These substituted formamidopyrimidine residues are actively removed from DNA by a DNA glycosylase present in E. coli cell extracts. The enz;me shows no apparent cofactor requirement and has a molecular weight of about 30 000. The release of ring-opened 7-methyl-guanine residues is due to a previously unrecognized activity, different from the three known E. coli DNA glycosylases that release uracil, 3-methyladenine, and hypoxanthine from DNA. This enzyme may serve to repair a major secondary alkylation product in DNA. In addition, it may remove nonmethylated purines, whose imidazole rings have been opened, from X-irradiated DNA.     

N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO₂(CH₂)₂-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents.     

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5′ cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn⁷GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N⁷-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5′-phosphorothioate (m⁷GMPS), which is a translation inhibitor with in vitro potency higher than bn⁷GMP.     

Oligonucleotides containing 7-vinyl-7-deazaguanine as a facile strategy for expanding the functional diversity of DNA

A modified nucleobase 7-vinyl-7-deazaguanine ((V)G) produced adducts with maleimides through Diels-Alder cycloaddition under very mild conditions. By this method, post-synthetic modification to oligonucleotides with diverse functionality (carboxylic acid, pyrene, benzophenone, succinimidyl ester, nitroxide and biotin) was accomplished.     

O6-methylguanine mutation and repair is nonuniform. Selection for DNA most interactive with O6-methylguanine

Mutations were induced in the ampicillinase gene of a bacteriophage f1/pBR322 chimera both by incorporation of O6-methyl-dGTP opposite T during DNA replication in vitro and by site-directed mutagenesis using O6-methylguanine-containing oligonucleotides. After passage of the DNA through Escherichia coli, analysis of 151 O6-methyl-dGTP-induced mutations indicated a significantly greater number of unmutated mutation sites than expected, whereas the mutated sites generally fit a Poisson distribution. The unmutated sites are assumed to be caused by the inability of some sequences to tolerate the presence of a tetrahedral methyl group within the confines of a Watson-Crick helix (Toorchen, D., and Topal, M.D. (1983) Carcinogenesis 4, 1591-1597). A consensus of the DNA sequences surrounding unmutated mutation sites was derived. The consensus sequence had significant similarity to the region of the rat Harvey ras oncogene containing the N-methyl-N-nitrosourea activated site for transformation (Zarbl, H., Sukumar, S., Arthur, A. V., Dionisio, M.-Z., and Barbacid, M. (1985) Nature 315, 382-385). We propose that direct alkylation at O6 of a guanine present within the consensus sequence may produce a DNA conformation less subject to repair. Mutation by O6-methylguanine-containing oligonucleotides demonstrated that repair of the O6-methylguanine lesions varied at least 3-4-fold with position of the lesion.     

Preparation and structural characterization of methylmercury(II) complexes of the minor tRNA-base 7-methylguanine

Methylmercury(II) complexes of 7-methylguanine (7mguaH) have been isolated from aqueous solution in the pH range 1-12 and structurally characterized. 1:1 complexes [(7mgua)HgCH₃]·2H₂O and [(7mguaH)HgCH₃][NO₃]· H₂O with respectively N1 - and N9-coordination (X-ray analyses) were obtained from solutions in the respective pH ranges 9–12 and 1–4. A 2:1 complex [(7mgua)(HgCH₃)₂][NO₃] with N1,N9-coordination (X-ray) may be prepared in the intermediate pH range 4–7. Two 3:1 complexes were isolated: [(7mgua)(HgCH₃)₃][NO₃]₂ from strongly acid solution (pH = 1–3), and [(7mguaH₋₁)(HgCH₃)₃][NO₃] in the pH range 7–9. Whereas an X-ray analysis establishes N1,N3,N9-coordination for the former species in the solid state, the ¹H NMR data suggest N2,N3,N9-coordination for the former and N2,N2,N9-coordination for the latter species in d₆-DMSO solution.