NONRANDOM DISTRIBUTION OF O6-METHYLGUANINE IN H-RAS GENE SEQUENCE FROM DNA MODIFIED WITH N-METHYL-N-NITROSOUREA

The distribution of O6-meG in the rat H-ras gene sequence was studied using PCR by transition of O6-meG to adenine during the reaction. In o rder to study the transition mutations the PCR product was cloned in a replicative form of phage M13mp18 and sequenced. The use of PCR for d etection of O6-meG was validated by using oligonucleotides (61 bases) containing one O6-meG residue at a defined site. After treatment of ra t liver DNA by N-methyl-N-nitrosourea in vitro, a striking nonrandom s equence distribution of O6-meG was observed. Sixty-eight per cent of O 6-methylated Gs were found in the middle G of the sequences GGT and GG A in the H-ras gene whereas no methylation was found in the middle G o f the sequences AGG, GGG, TGT, TGC, CGA and CGC. No O6-meG adduct was found in the 12th codon of H-ras (sequence GGA). The frequency of O6-m eG formation as a function of two flanking nucleotides on each side of the target guanine was calculated as an approach to understanding mor e distant sequence effects. It was found that in the DNA sequence stud ied the formation of O6-meG was highest if the G was flanked by PyPu o r PuPu on the 5' side (Py, pyrimidine and Pu, purine) whereas PuPu on the 3' side showed maximal inhibition of O6-meG formation.