INHIBITION OF CLEAVAGE BY RESTRICTION ENDONUCLEASES DUE TO MODIFICATIONS INDUCED IN SV40 DNA BY METHYL METHANESULFONATE

Methyl methanesulfonate (MMS), a direct mutagen, methylates DNA bases and causes distortions in DNA structure. Supercoiled SV40 DNA was trea ted in vitro with varying concentrations of MMS from 0.001 mM to 10 mM MMS either for 30 min or 3 h and analysed by electrophoresis in 1% ne utral and alkaline agarose gels. The electrophoretic mobility (EPM) of native DNA did not change after treatment with the mutagen, while alk aline gels revealed low MW DNA fragments due to single strand breaks a t alkali-sensitive sites generated by the action of MMS. By two-dimens ional electrophoresis, we find that all three native DNA forms contain alkali-sensitive sites after treatment with MMS. To examine the effec t of base modification by MMS on DNA-protein interactions, we have use d as probes, restriction endonucleases. These cleave DNA in a sequence -specific manner, and their activity is dependent upon the methylation status of the substrate DNA. We find that cleavage by these restricti on endonucleases is inhibited due to methylation by MMS.