Ring-opened 7-methylguanine nucleotides are resistant to nuclease P1 digestion and good substrates to polynucleotide kinase

Abstract

Dimethyl sulfate was used to prepare 7-methyl-2′-deoxy-guanosine 3′-monophosphate (7-methyl-dGMP), whkh was ring-opened in alkali to 2′-deoxy-N⁵-methyl-N⁵-formyl-2,5,6-triamino4-oxopyrimidine 3′-monophosphate (ROM-dGMP). ROM-dGMP was not dephosphorylated by nuclease P₁ in contrast to normal deoxynucleotides. It was efficiently 5′-phosphorylated by T₄ polynucleotide kinase. When methylated DNA was alkali-treated and digested with mkro-coccal nuclease, spleen phospbodiesterase and nuclease P₁, ROM-dGMP was formed and this was labeled with [_γ-³²P]-ATP hi the presence of polynucleotide kinase. Ring-opening and P₁ treatment appear methods of choice for ³²P-post-labeUng of 7-alkylguanines in DNA.