32P-Postlabeling of N-(deoxyguanosin-8-yl)arylamine adducts: a comparative study of labeling efficiencies.
32P-Postlabeling is an extremely powerful technique for the detection of DNA adducts. Typically, the quantitation of DNA adducts by (32)P-postlabeling is achieved by relative adduct labeling, via comparison of the radioactivity incorporated into the adducts to that associated with the normal nucleotides. This approach is based on a number of assumptions, the foremost being that normal and adducted nucleotide 3'-phosphates are converted to 3', 5'-bisphosphates with similar efficiencies. To evaluate labeling efficiencies for specific DNA adducts, we conducted a comparative study of the kinetics of phosphorylation by T(4) polynucleotide kinase using 2'-deoxyguanosine 3'-phosphate (dG3'p) and a series of N-(deoxyguanosin-8-yl)arylamine 3'-phosphate adduct standards (dG3'p-C8-Ar, Ar being 4-aminobiphenyl, 3- and 4-methylaniline, and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3'p and the dG3'p-C8-Ar adducts followed Michaelis-Menten kinetics. The apparent turnover numbers were 40-240-fold lower when labeling dG3'p-C8-Ar adducts compared to that when labeling dG3'p. The apparent specificity constant calculated for dG3'p-C8-4-aminobiphenyl (1.4 microM(-)(1) min(-)(1)) was approximately 4-fold lower than that (5. 4 microM(-)(1) min(-)(1)) found for dG3'p. Apparent specificity constants for the monoarylamine adducts were even lower (0.043-0.23 microM(-)(1) min(-)(1)) and decreased in the following order: 4-methylaniline > 3,4-dimethylaniline > 3-methylaniline > 2, 4-dimethylaniline. Similar experiments conducted with dG3'p-C8-Ar standards for 2-methylaniline and 2,3-dimethylaniline produced very poor and irreproducible labeling. These results indicate that (32)P-postlabeling of dG3'p-C8-Ar adducts is less efficient than that of dG3'p and suggest that normal nucleotides will be labeled preferentially to the arylamine adducts under kinetically controlled conditions. The data also indicate a further decrease in labeling efficiency upon substitution ortho to the amino group (e.g., 2, 4-dimethylaniline). In addition, the ATP concentrations required for optimal labeling were found to be substantially higher than those used in typical (32)P-postlabeling assays. Since the high specific activity of carrier-free [gamma-(32)P]ATP precludes increasing the ATP concentration to a significant extent, these data emphasize the need for using highly efficient adduct enrichment procedures when conducting (32)P-postlabeling analyses of DNA adducts.
Mourato
L L
LL
Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Beland
F A
FA
Marques
M M
MM
D000255:Adenosine Triphosphate / Q000737:chemistry; D000611:Aminobiphenyl Compounds / Q000737:chemistry*; D000814:Aniline Compounds / Q000737:chemistry*; D002855:Chromatography, Thin Layer; D018736:DNA Adducts / Q000737:chemistry*; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry*; D007553:Isotope Labeling; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry
D000611:Aminobiphenyl Compounds; D000814:Aniline Compounds; D018736:DNA Adducts; D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; C034009:2'-deoxyguanosine 3'-phosphate; D000255:Adenosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D003849:Deoxyguanosine