Quantitative and qualitative analysis of DNA methylation at N-3-adenine byN-methyl-N-nitrosourea

The sequence-specific alkylation of DNA by N-methyl-N-nitrosourea (MNU) has been demonstrated for the minor groove N-3-methyladenine (N-3-MeAde) adduc t using neutral thermal hydrolysis and polyacrylamide sequencing gels. The ratio of relative yields of N-7- and N-3-MeAde and N-7-methylguanine (N-7-M eGua) is approximately 0.03:0.15:1.00, respectively, on the basis of the ge l data, and these values are comparable to relative yields determined by bu lk digestion of MNU-methylated DNA when HPLC was used to analyze the indivi dual adducts, In contrast to the methylation at N-7-guanine (N-7-Gua) by MN U, alkylation at Ade shows minimal sequence selectivity. Similar to the met hylation at N-7-Gua, formation of N-3-MeAde by MNU is inhibited by 50-200 m M concentrations of NaCl and DNA binding cations, including distamycin and spermine. However, N-3-MeAde formation at Ade residues within methidiumprop yl-EDTA-Fe(II) footprinted distamycin DNA affinity binding regions is selec tively inhibited at low concentrations of distamycin relative to Ade sites outside of ligand binding regions, and N-7-Gua within or outside the distam ycin binding regions. HPLC analysis shows that distamycin also quantitative ly inhibits the production of N-3-methylguanine when calf thymus DNA is tre ated with MNU or methyl methanesulfonate. The specific inhibitory effect of distamycin, which binds in the minor groove at Ade/Thy-rich sequences, pro vides additional evidence that the predominant DNA lesion detected at Ade b y sequencing gel analysis involves minor groove N-3-MeAde modifications.