HIGH-RESOLUTION TLC MAPPING AND CHARACTERIZATION OF P-32 POSTLABELED MONOPHOSPHATE 7,12-DIMETHYLBENZ[A]ANTHRACENE DNA ADDUCTS

Previous work has shown that 7,12-dimethylbenz [a]anthracene (DMBA)-DNA adducts can be converted by 32P-post-labeling to different types of radiolabeled derivatives (nucleoside 3',5'-bisphosphates, nucleoside 5'-monophosphates and dinucleotides), and that the 32 3',5'-bisphosphate derivatives can be further characterized by cross-referencing with 3H-labeled nucleoside DMBA adducts, for which structural information is available. This work has now been extended by TLC comparisons of 5'-monophosphate and 3',5'-bisphosphate DMBA adducts. To this end, DMBA modified DNA was enzymatically hydrolyzed to 3'-mono-phosphates (Xp+Np) or to dinucleotides (XpN), and these digestion products were 32P-postlabeled by published procedures to yield 3',5'-bisphosphate (*pXp) or 5'-monophosphate (*pX) adducts. Individual *pXp and *pX fractious were isolated from polyethyleneimine (PEI)-cellulose TLC maps and chromatographically compared after enzymatic 3'-dephosphorylations of the 3',5'-bisphosphate (*pXp) derivatives (*pXp→*pX+Pi) Four reactions were standardized and employed for this purpose: (i) 3'-dephos-phorylation by extensive digestion with nuclease P1; (II) 3'-dephosphorylation catalyzed by polynucleotide kinase; (iii) partial dephosphorylation by bacterial alkaline phosphatase; and (iv) partial dephosphorylation by prostatic acid phos phatase. Individual DMBA adducts displayed marked differences with regard to their susceptibility to enzymatic dephosphorylation. The three major and most minor post-labeled 5'-monophosphate DMBA adducts were cross-referenced this way with 3',5'-bisphosphate and nucleoside adducts, so that specific dihydrodiol epoxide-nucleoside 5'-monophosphate adducts can now be identified and measured by 32P-postlabeling. © 1990 Oxford University Press.