The mechanism of the mutagenic action of hydroxylamine XII. Phenotypic suppression of amber mutants of phage T7 by hydroxylamine and O-methylhydroxylamine.

The reproduction of phage T7 in the presence of hydroxylamine (HA) (mutagenesis in vivo) results in the phenotypic suppression of some amber mutants. The presence of O-methylhydroxylamine (OMHA) results in a similar effect, indicating a similar mechanism for the action of the two compounds. Since the rate of reaction of mutagen with nucleoside residues under these conditions in negligibly low, one of the most plausible explanations of this effect is the enzymic formation of modified precursors and their incorporation into bacterial tRNAs or phage-induced RNA.     

Induction of intragenic mitotic recombination in yeast by hydroxylamine and its mutagenic specificity

Summary Hydroxylamine in 1 M concentration is capable of inducing mutations (Table 1) and recombination (Figs. 1–3) in yeast. It is postulated that HA-produced lesions in DNA are repaired and thus HA-induced mutations in yeast cannot be specific with respect to the type of base substitution; the recombinogenic ability of a mutagen can be considered as an indication that the lesions in DNA produced by this mutagen are repaired.     

Mechanism of the mutagenic action of hydroxylamine. IX. The UV-induced cleavage of the N-O bond in N4-hydroxy-and N4-methoxycytidine and N6-methoxyadenosine.

The principal UV-induced (lambda = 2546nm) reaction of N4-hydroxy- and N4methoxycytidines and N6-methoxyadenosine in neutral aqueous solutions is cleavage of the exocyclic N-O bond with the respective formation of cytidine and adenosine. Quantum yields are 2.8x10(-3) and 2.2x10(-3) M/E for the first two compounds and 9.1x10(-3) M/E for N6-methoxyadenosine.     

Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.     

Mechanism of potent mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine on intracellular phage lambda.

N-Methyl-N′-nitro-N-nitrosoguanidine efficiently induces mutations from “clear” to “virulent” in phage λ only during the intracellular growth phase. Lambda DNA extracted from infected bacteria after treatment with MNNG³ produced a mutant yield about 100-fold higher than the spontaneous level upon transfection of MNNG-treated spheroplast cells, whereas the yield diminished an order of magnitude when assayed on untreated spheroplasts. As measured by ¹⁴C incorporation after treatment with [methyl-¹⁴C]MNNG, λ DNA packed in head protein was methylated to about 3% by an MNNG dose of 0.6 mg/ml but was barely mutagenised; whereas intracellular λ DNA was methylated to no more than 0.6% by an MNNG dose of 0.09 mg/ml and was highly mutagenised. Lambda phages treated in vitro with ethyl methanesulfonate produced a rather low mutant yield on untreated cells but the yield increased about tenfold on MNNG-treated cells. Mutability of untreated λ on cells having received an F′ factor was enhanced efficiently by ultraviolet light, but not so by MNNG, previously applied to the F′. Surprisingly similar MNNG dose-effect curves exist for enhancing spontaneous, mispairing (MNNG or EMS induced) and misrepair (ultraviolet light induced) mutagenesis of λ. From these and other data we conclude that MNNG hypermutagenesis results from a synergistic increase in mispairing probability of appropriately methylated bases (by action of MNNG in vivo) in the target gene within an MNNG-induced intracellular environment that has an enhanced mutagenic capacity.     

The mechanism of the mutagenic action of hydroxylamine XIII. Reversion of phage MS2 amber mutants in the presence of hydroxylamines.

The replication of the phage MS2 in the presence of either hydroxylamine (HA) or O-methylhydroxylamine (OMHA) (mutagenesis in vivo) results in an increase in the reversion frequency of two amber mutations in the maturation protein. When acting on the extracellular phage (mutagenesis in vitro) the mutagens do not affect the reversion frequency. The most probable mode of mutagenic action of the hydroxylamines on the vegetative MS2 phage involves the enzymic formation of modified precursors and their incorporation into RNA.     

The mutagenic action of nitroimidazoles. IV. A comparison of the mutagenic action of several nitroimidazoles and some imidazoles.

The mutagenic action of 51 imidazoles was investigated. The fluctuation test of Luria and Delbrück was used, with Klebsiella pneumoniae as test organism. 8 compounds, including 5 with a weak mutagenic action in the fluctuation test, were also investigated by the Ames test in which Salmonella typhimurium TA100 was used. Of the 51 imidazoles examined, 33 were nitroimidazoles. 31 of the latter appeared to be mutagenic, whereas out of the 18 other imidazoles without a nitro group only 2 were mutagenic. Several of the substances tested for mutagenicity showed an antimicrobial activity. No direct relationship between antimicrobial action, growth inhibition and mutagenicity was established. With methyl-nitroimidazoles a relationship was found between the chemical structure and mutagenic action. However, when the nitroimidazoles had a more complex chemical structure, a relationship between this structure and mutagenicity could not be established.