This paper describes the application of the novel nonradioactive technique for studying the sequence selectivity of selected alkylating agents. N-Nitroso-N-methylurea (MNU) and N-methyl-N'-nitro-nitrosoguanidine (MNNG) were chosen from the SN1 group of alkylating agents. Dimethyl sulphate (DMS) was used to represent alkylation profile produced by the SN2 compounds. Results of SN1 compounds indicated that in a run (G)3 the latter two Gs are more susceptible to alkylation than the most 5' G. Moreover, in a GG sequence the 3' G seems to be more alkylated. This effect is more evident when the GG site was preceded by a 5' pyrimidine. These findings suggest that a regio-selective mechanism, rather than the formation of diazonium ions, accounts for DNA alkylation by SN1 compounds. On the other hand, DMS showed preferential alkylation of the 5' end in a (G)3 run. However, at GG sequences no clear preferred site of alkylation could be distinguished. Lack of specificity of SN2 compound would seem to suggest that other factors as well as the primary DNA structure may play a role in determining the extent of alkylation at a certain site.
We have developed a method to determine rapidly the sequence specificity of DNA alkylation resulting from chemical treatment. The utility of this approach is demonstrated here in a study of the sequence specificity of alkylation by dimethylsulphate (DMS). The method is independent of the sequence chosen and makes use of the polymerase chain reaction (PCR) to generate a fluorescently labelled DNA target. In this study, a 302 bp segment of the Escherichia coli lacI gene was amplified and the product purified by liquid chromatography on a Mono Q column. This DNA was alkylated with DMS and treated with hot piperidine to produce single-strand breaks at sites of N7 alkylation. The distribution of the break points, and hence the position and extent of alkylation, were determined on an Applied Biosystems 370A automated DNA sequencer.
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