A new sensitive 32P-postlabeling assay based on the specific enzymatic conversion of bulky DNA lesions to radiolabeled dinucleotides and nucleoside 5'-monophosphates

Abstract

A new sensitive ³²P-postlabeling assay for DNA adducts has been developed in which DNA is hydrolyzed initially by nuclease P1 and prostatic acid phosphatase instead of micrococcal nuclease and spleen phosphodiesterase as employed in previous postlabeling procedures. When DNA containing bulky adducts, X₁, X₂,.....X_n, is digested with nuclease P1 at pH 5, normal nucleotides are released as 5'-monophosphates, pN, while adducts are excised as 5'-phosphorylated dinucleotides, pX₁pN, because internucleotide linkages on the 3' side of X resist attack by nuclease P1. Addition of prostatic acid phosphatase to such a digest results in 5'-dephosphorylation of the nucleotides to normal nucleosides, N, and adducted dinucleotides, X₁pN, carrying a 5'-terminal free hydroxyl group. The dinucleotides but not nucleosides are converted to 5'-³²P-labeled dinucleotides, [³²P]pX₁pN, by T4 polynucleotide kinase-catalyzed [³²P]phosphate transfer from [γ-³²P]ATP. Upon mapping on polyethyleneimine-cellulose anion-exchange TLC, the labeled dinucleotide adducts produce characteristic autoradiographic fingerprints. Alternatively, they are further digested with snake venom phosphodiesterase to yield 5'-monophosphates, (³²P)pX₁ and pN. TLC profiles of the monophosphate adducts are distinct from those of the dinucleotides. These reactions provide the basis of the new ³²P-postlabeling scheme, which is compared in this paper with a previously reported protocol yielding adducts in the form of 5'-³²P-labeled 3', 5'-bisphosphates, [³²P]pX₁p. The results show that the availability of three different types of ³²P-postlabeled derivatives for the same adduct aids in the analysis and chromatographic characterization of DNA adducts from diverse exogenous and endogenous sources.