Hydroxylamine and methoxyamine mutagenesis: displacement of the tautomeric equilibrium of the promutagen N6-methoxyadenosine by complementary base pairing (original) (raw)
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Biophysical Chemistry, 1993
A study of the interaction of N6-methoxyadenosine (OMe6A, which exists as an equilibrium mixture of amino and imino tautomers) with the potentially complementary uridine in non-aqueous medium demonstrated that only the tautomer amino-0Me6A base pairs with uridine. The association constant, determined by 'H NMR spectroscopy, was 101 Mm' at +3o"C, an order of magnitude greater than those for autoassociation of amino-0Me6A or uridine. Base pair formation between amino-0Me6A and uridine led to a shift of the amino-imino equilibrium of 10% in favour of the amino species which associates with uridine, with the concomitant decrease in population of the imino form. Base pairing was accompanied by intermolecular proton exchange between the N6-H of amino-OMe'A and the N(3)-H of uridine. The rate constants for these exchanges, as well as for tautomeric exchange of OMe6A in the presence of uridine, were measured by means of the saturation transfer technique. The mechanism of proton exchange is compared with, and shown to be different from, that previously observed for base pair formation between the imino tautomer of 0Me6A and cytidine (Stolarski et al., Biochemistry 26 (19871, 4332 also linked with tautomeric exchange. The resulting proposed schemes of base pairing are dependent on the conformation of the exocyclic N6-methoxy group, and are compared and discussed with reference to published data on base pairing at the oligonucleotide duplex level, as well as known data on base pairing of the analogous promutagen N4-methoxycytidine. The overall findings further contribute to our understanding of the dual functionality of the promutagen OMe6A at the molecular level, and furthermore, provide an excellent model system for studying the role of tautomerism on proton exchange in base pairs.
Base pairing-induced shift in tautomeric equilibrium of a promutagenic analogue, N6-methoxyadenosine
FEBS Letters, 1983
The nuclear magnetic resonance spectra of N6-methoiyadenosine and of uridine, both methylated in the 2'-, 3'-and 5'-positions to obtain solution in deuterochloroform, reveal the formation of hetero-associates in which the amino-tautomeric equilibrium is shifted to the amino form. These results ar discussed in terms of the mutagenicity of 0-methylhydroxylamine which converts adenosine to N6-methoxyadenosine.
Proceedings of the National Academy of Sciences, 1986
2-Aminopurine (AP), a potent mutagenic base analogue, most frequently pairs with thymine. In the APT base pair, both bases adopt normal tautomeric forms. The mechanism for the mutagenic activity arises from its observed pairing with cytosine, which has been ascribed to an enhanced tendency to adopt the rare imino tautomeric form. NMR studies in H20 on all the exchangeable protons in an oligonucleotide duplex containing an APT base pair show Watson-Crick hydrogen bonding. When the thymine is replaced by cytosine in the duplex, we observe an AP-C base pair. Both amino protons of AP are seen excluding the rare tautomeric form. Although several alternative structures are possible, it is shown that the second hydrogen bond is formed by protonation of the AP-C base pair and that this is the dominant species under physiological conditions.
Mutagenicity Testing of 9-N-Substituted Adenines and Their N-Oxidation Products
Environmental Health Perspectives, 1993
Adenine together with certain 9-N-substituted derivatives such as 9-methyl, 9-benzyl, 9-benzhydryl, and 9-trityl were tested against Salmonella typhimurium strains TA97, TA98, and TA100 in the absence and presence of rat hepatic S9 prepared from Aroclor 1254 pretreated rats. All compounds were positive toward TA98 in the presence of the metabolic activating system, whereas they all lacked mutagenic activity in the absence of S9, and toward TA97 and TA100 with or without S9 when tested at 100 ng/plate. A similar pattern was observed for the corresponding 1-N-oxides. 6-Hydroxylaminopurine was not mutagenic toward TA100 at 100 ng/ plate, whereas it was toxic toward TA97 and TA98 at this level. When tested at 1 ng/plate, hydroxylaminopurine was still toxic to TA98 but produced twice the spontaneous reversion rate to TA97 without metabolic activation. Surprisingly, 9-methyl-6-hydroxylaminopurine was only active toward TA98 in the presence of S9, whereas 9-benzyl-6-hydroxylaminopurine was highly active toward TA97 and TA100 in the absence of S9 and even more active in the presence of S9. This compound was inactive toward TA98 in the absence of S9. The results generally support the concept that nuclear N-oxidation of aminoazaheterocycles is a detoxication process, whereas N-hydroxylation of the exo amino group is a toxication reaction.
Nucleic Acids Research, 1998
The structure of the deoxyribonucleoside derived from N 6 -methoxy-2,6-diaminopurine (dK) was examined by NMR. The methoxyamino residue was found predominantly in the imino rather than the amino tautomer (ratio: 9:1 in DMSO). The nucleoside proved to be a potent transition mutagen in Escherichia coli, in contrast to the closely related nucleoside derived from the analogue N 6 -methoxyaminopurine (dZ), which was only weakly mutagenic. The 5′-triphosphate derivatives, dKTP and dZTP, were synthesized; Taq polymerase incorporated dKTP opposite both T and, less well, opposite dC in template DNA. Both analogue triphosphates produced transition mutations when added to PCR reactions. In each case, there was a large excess of AT→GC compared to GC→AT mutations (ratios were 15:1 for dKTP and 10:1 for dZTP). Polymerase extension times in each cycle had to be extended, consistent with a decreased rate of DNA synthesis in the presence of the analogues. This and the mutagenic ratios are discussed in terms of syn-anti inversion of the methoxyl group.
Chemical Research in Toxicology, 2002
8-Oxoguanine is a mutagenic oxidative damage product of guanine that has been the subject of many experimental studies. Despite numerous references to this damaged base, its precise configuration or population of configurations in equilibrium are unknown, as it can be drawn in over 100 potential neutral and ionized tautomeric forms. The structural uncertainty surrounding 8-oxoguanine complicates mechanistic studies of its mutagenicity and capacity to be recognized for repair. Experimental measurements on the tautomeric equilibria and pK a values of 8-oxoguanine are complicated by its insolubility in water. Therefore, we used first principles quantum mechanics (density functional theory, B3LYP, in combination with the Poisson-Boltzmann continuum-solvation model) to investigate the relative stabilities and sitespecific pK a values of various neutral and ionized tautomers of 8-oxoguanine. We show that the major tautomer of neutral 8-oxoguanine in aqueous solution is the 6,8-diketo form 2, and that 8-oxoguanine has increased acidity at N1 relative to guanine. Our calculations on 2′deoxyguanosine-3′,5′-bisphosphate and its 8-oxo analogue support the accepted conclusion that repulsion between the O8 of 8-oxoguanine and O5′ of the backbone sugar promote 8-oxoguanine: adenine pairings in the syn:anti conformation. Further, we show that the N7 proton of 8-oxoguanine is difficult to remove either through tautomerization or ionization, consistent with its involvement as an important landmark in distinguishing guanine from 8-oxoguanine. The possibility of additional structural landmarks that distinguish 8-oxoguanine from guanine, and a possible mechanism for glycosylase removal of 8-oxoguanine are discussed.