Quantitative and kinetic examination of 32P-postlabeling of etheno-substituted nucleotides

1,N6-ethenodeoxyadenosine-, 1,N2-ethenodeoxyguanosine- and 3,N4-ethenodeoxycytidine-3'-monophosphates were labeled by [gamma-32P] ATP using T4 polynucleotide kinase in conditions commonly used for the 32P-postlabeling assay. Kinetic studies showed that the reaction is fast reaching a plateau after 15-30 min. The efficiency of phosphorylation, as studied by substrate-product concentration dependency, was between 50-100% at the lower substrate concentrations. The adducts are labeled efficiently at sub-femtomole levels. All the adducts were sensitive to the 3'-dephosphorylation by P1 nuclease although the guanine derivative appeared to be more resistant than the two other adducts.