Randerath K, Gupta K P, van Golen K L
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Carcinogenesis. 1993 Aug;14(8):1523-9. doi: 10.1093/carcin/14.8.1523.
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.
当通过³²P后标记法的双磷酸酯版本分析时，被香料成分黄樟素（1-烯丙基-3,4-亚甲二氧基苯）的代谢物加合的小鼠肝脏DNA呈现出两种主要加合物，它们先前已被鉴定为N²-（反式异黄樟素-3'-基）2'-脱氧鸟苷3',5'-双磷酸酯（加合物1）和N²-（黄樟素-1'-基）2'-脱氧鸟苷3',5'-双磷酸酯（加合物2）。然而，通过该检测方法的二核苷酸/单磷酸酯版本对相同的DNA制剂进行分析时，在聚乙烯亚胺纤维素薄层层析（PEI-纤维素TLC）上出现了另外两个斑点，其性质在本研究中得以阐明。利用了几种酶（T4多核苷酸激酶、核酸酶P1、蛇毒磷酸二酯酶和脾脏磷酸二酯酶）来水解这些化合物，并且产物在PEI-纤维素薄层上与已知结构的放射性标记和非放射性核苷酸进行共层析。发现另外的斑点是在5'-和3'-羟基处都带有³²P标记的加合二核苷酸。T4多核苷酸激酶催化的3'-磷酸化具有高度特异性，因为只有在3'-位置具有未修饰嘌呤的加合物1的二核苷单磷酸衍生物才易受该酶的5'-和3'-磷酸化作用。因此，另外两种³²P标记的黄樟素衍生物的结构为pX1pAp和pX1pGp，其中X1表示N²-（反式异黄樟素-3'-基）2'-脱氧鸟苷。T4多核苷酸激酶的官方名称，即ATP：5'-去磷酸多核苷酸5'-磷酸转移酶（EC 2.7.1.78），表明了该酶作为5'-磷酸激酶的特定作用。尽管该酶在酸性pH下具有3'-磷酸酶活性，但此前尚未报道有3'-激酶反应。讨论了致癌物-DNA加合物能够特异性改变蛋白质-核苷酸相互作用保真度这一发现对化学致癌作用的可能影响。
