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 safrole (1-allyl-3,4-methylenedioxybenzene), when analyzed via the bisphosphate version of the 32P-postlabeling assay, exhibits two major adducts, 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 nature was clarified in the present study. Several enzymes (T4 polynucleotide kinase, nuclease P1, venom phosphodiesterase and spleen phosphodiesterase) were utilized to hydrolyze these compounds, and the products co-chromatographed on PEI-cellulose thin layers with radiolabeled and non-radioactive nucleotides of known structure. The additional spots were found to be adducted dinucleotides carrying 32P-label at both the 5'- and 3'-hydroxyls. T4 polynucleotide kinase-catalyzed 3'-phosphorylation was highly specific in that only dinucleoside monophosphate derivatives of adduct 1, with an unmodified purine in the 3'-position, were susceptible to both 5'- and 3'-phosphorylation by the enzyme. Thus, the structures of the two additional 32P-labeled safrole derivatives were pX1pAp and pX1pGp where X1 denotes N2-(trans-isosafrol-3'-yl)2'-deoxyguanosine. The official name of T4 polynucleotide kinase, ATP:5'-dephosphopolynucleotide 5'-phosphotransferase (EC 2.7.1.78), denotes the specific action of this enzyme as a 5'-phosphokinase. Although the enzyme has 3'-phosphatase activity at acidic pH, no 3'-kinase reaction has been previously reported. Possible implications for chemical carcinogenesis of the finding that carcinogen-DNA adducts can specifically alter the fidelity of protein-nucleotide interactions are discussed.
Randerath
K
K
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Gupta
K P
KP
van Golen
K L
KL
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology*; D002845:Chromatography; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D005260:Female; D006868:Hydrolysis; D051379:Mice; D008815:Mice, Inbred Strains; D009710:Nucleotide Mapping; D009711:Nucleotides / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism*; D011854:Radiochemistry; D012451:Safrole / Q000378:metabolism / Q000633:toxicity*; D012680:Sensitivity and Specificity; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013154:Spleen / Q000201:enzymology
D009711:Nucleotides; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010727:Phosphoric Diester Hydrolases; D012451:Safrole