Ring-opened 7-methylguanine nucleotides are resistant to nuclease P1 digestion and good substrates to polynucleotide kinase.
Dimethyl sulfate was used to prepare 7-methyl-2'-deoxy-guanosine 3'-monophosphate (7-methyl-dGMP), which was ring-opened in alkali to 2'-deoxy-N5-methyl-N5-formyl-2,5,6-triamino-4-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 micrococcal nuclease, spleen phosphodiesterase and nuclease P1, ROM-dGMP was formed and this was labeled with [gamma-32P]-ATP in the presence of polynucleotide kinase. Ring-opening and P1 treatment appear methods of choice for 32P-post-labeling of 7-alkylguanines in DNA.
Hemminki
K
K
Institute of Occupational Health, Helsinki, Finland.
D003848:Deoxyguanine Nucleotides / Q000138:chemical synthesis*; D008836:Micrococcal Nuclease / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology
D003848:Deoxyguanine Nucleotides; C060943:7-methyl-2'-deoxyguanosine 3'-monophosphate; C060945:2'-deoxy-N(5)-methyl-N(5)-formyl-2,5,6-triamino-4-oxopyrimidine 3'-monophosphate; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D009708:Nucleotidases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008836:Micrococcal Nuclease; D010727:Phosphoric Diester Hydrolases