32P-postlabeling of 7-methylguanine adducts in DNA.
The 32P-postlabeling technique introduced by Randerath and co-workers has been particularly successful with stable and nonpolar DNA adducts, but the assay has not been used to any large extent to detect 7-alkylguanine derivatives. In the present communication, we have investigated the phosphorylation reaction by T4 polynucleotide kinase using 7-methyl-3'-dGMP, ring-opened 7-methyl-3'-dGMP and enzyme-digested methylated DNA as substrates. The methylated substrates were detected at femtomol (fmol) sensitivities. 7-methyl-3'-dGMP was quantitatively phosphorylated at these low concentrations. The efficiency of phosphorylation of the ring-opened product was less. It was shown that ring-opened 7-methyl-3'-dGMP was resistant to digestion with nuclease P1, making alkali-treatment and enzyme digestion of DNA possible approaches to the determination of 7-methylguanine in DNA.
Hemminki
K
K
Institute of Occupational Health, Helsinki, Finland.
Mustonen
R
R
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology; D008745:Methylation; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism
D006147:Guanine; C008450:7-methylguanine; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase