ALTERED FIDELITY OF A NUCLEIC-ACID MODIFYING ENZYME, T4 POLYNUCLEOTIDE KINASE, BY SAFROLE-INDUCED DNA-DAMAGE

Mouse liver DNA adducted with metabolites of the spice constituent saf role (1-allyl-3,4-methylenedioxybenzene), when analyzed via the bispho sphate version of the P-32-postlabeling assay, exhibits two major addu cts, which had been previously identified as N2-(trans-isosafrol-3'-yl )2'-deoxyguanosine 3',5'-bisphosphate (adduct 1) and N2-(safrol-1'-yl) 2'-deoxyguanosine 3',5'-bisphosphate (adduct 2). However, analysis of the same DNA preparation by the dinucleotide/monophosphate version of the assay gave two additional spots on PEI - cellulose TLC whose natur e was clarified in the present study. Several enzymes (T4 polynucleoti de kinase, nuclease P1, venom phosphodiesterase and spleen phosphodies terase) were utilized to hydrolyze these compounds, and the products c o-chromatographed on PEI-cellulose thin layers with radiolabeled and n on-radioactive nucleotides of known structure. The additional spots we re found to be adducted dinucleotides carrying P-32-label at both the 5'- and 3'-hydroxyls. T4 polynucleotide kinase-catalyzed 3'-phosphoryl ation was highly specific in that only dinucleoside monophosphate deri vatives of adduct 1, with an unmodified purine in the 3'-position, wer e susceptible to both 5'- and 3'-phosphorylation by the enzyme. Thus, the structures of the two additional P-32-labeled safrole derivatives were pX1pAp and pX1pGp where X1 denotes N2-(trans-isosafrol-3'-yl)2'-d eoxyguanosine. The official name of T4 polynucleotide kinase, ATP:5'-d ephosphopolynucleotide 5'-phosphotransferase (EC 2.7.1.78), denotes th e specific action of this enzyme as a 5'-phosphokinase. Although the e nzyme has 3'-phosphatase activity at acidic pH, no 3'-kinase reaction has been previously reported. Possible implications for chemical carci nogenesis of the finding that carcinogen - DNA adducts can specificall y alter the fidelity of protein-nucleotide interactions are discussed.