32P‐postlabeling of 7‐methylguanine adducts in DNA

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Abstract
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‐methyl‐guanine in DNA.

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