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.