A Postlabeling Assay for Oxidative Damage

  • Michael Weinfeld
  • Michel Liuzzi
  • George D. D. Jones


The 32P-postlabeling assay was originally devised by Randerath et al. (1981) to measure carcinogen-DNA adducts. In the procedure, DNA is first digested by micrococcal nuclease and calf spleen phosphodiesterase to give nucleoside 3′-monophosphates (normal and modified) that are subsequently labeled by incubation with [γ-32P]-ATP and T4 polynucleotide kinase. The radiolabeled compounds are then separated by two-dimensional TLC. The assay has two important advantages. First, there is no requirement for prelabeling the DNA, which makes the assay useful for the study of DNA lesions in tissues. Second, because of the availability of [γ-32P]-ATP of high specific activity, the assay permits detection at the femtomole level. There are, however, two major drawbacks:(1) the polynucleotide kinase must be able to act on the modified nucleoside 3′-monophosphate and (2) the resulting labeled modified nucleoside diphosphate must be separable from the high background of normal nucleoside diphosphates. These problems are well illustrated in the reports of efforts to detect thymine glycols, a well-known oxidative base lesion, in irradiated DNA (Reddy et al., 1991; Hegi et al., 1989). Furthermore, lesions that involve base loss, such as abasic sites and deoxyribose fragments, cannot be detected by this approach.


Abasic Site Micrococcal Nuclease Modify Nucleoside Radioactive Band Buffer Chamber 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Baleja, J. D., Buchko, G. W., Weinfeld, M., and Sykes, B. D. (1993). Characterization of γ-radiation induced decomposition products of thymidine-containing dinucleoside monophosphates by NMR spectroscopy. J. Biomol. Struct. Dynam. 10:747–762.CrossRefGoogle Scholar
  2. Bertoncini, C. R. A., and Meneghini, R. (1995). DNA strand breaks produced by oxidative stress in mammalian cells exhibit 3′-phosphoglycolate termini. Nucleic Acids Res. 23:2995–3002.PubMedCrossRefGoogle Scholar
  3. Buchko, G. W., and Weinfeld, M. (1993). Influence of nitrogen, oxygen, and nitroimidazole radiosensitizers on DNA damage induced by ionizing radiation. Biochemistry 32:2186–2193.PubMedCrossRefGoogle Scholar
  4. Bykov, V. J., Kumar, R., Försti, A., and Hemminki, K. (1995). Analysis of UV-induced DNA photoproducts by 32P-postlabelling. Carcinogenesis 16:113–118.PubMedCrossRefGoogle Scholar
  5. Försti, A., and Hemminki, K. (1994). A 32P-postlabelling assay for DNA adducts induced by cis-diammine-dichloroplatinum(II). Cancer Lett. 83:129–137.PubMedCrossRefGoogle Scholar
  6. Gillardeaux, O., Périn-Roussel, O., Nocentini, S., and Périn, F. (1994). Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells. Carcinogenesis 15:89–93.PubMedCrossRefGoogle Scholar
  7. Harley, C. B., and Vaziri, H. (1991). Deproteinization of nucleic acids by filtration through a hydrophobic membrane. GATA 8:124–128.Google Scholar
  8. Hegi, M. E., Sagelsdorff, P., and Lutz, W. (1989). Detection by 32P-postlabeling of thymidine glycol in γ-irradiated DNA. Carcinogenesis 10:43–47.PubMedCrossRefGoogle Scholar
  9. Hemminki, K., Försti, A., Löfgren, M., Segerbäck, D., Vaca, C, and Vodicka, P. (1993). Testing of quantitative parameters in the 32P-postlabelling method, in:Postlabelling Methods for the Detection of DNA Damage, IARC Scientific Publications No. 124 (D. H. Phillips, M. Castegnaro, and H. Bartsch, eds.), IARC Publications, Lyon, pp. 51–63.Google Scholar
  10. Henner, W. D., Rodriguez, L. O., Hecht, S. M., and Haseltine, W. A. (1983). γ ray induced deoxyribonucleic acid strand breaks. J. Biol. Chem. 258:711–713.PubMedGoogle Scholar
  11. Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989). Enzymatic analysis of isomeric trithymidylates containing UV light-induced cyclobutane pyrimidine dimers: Nuclease Pl-mediated hydrolysis of the intradimer phosphodiester linkage. J. Biol. Chem. 264:6355–6363.PubMedGoogle Scholar
  12. Randerath, K., Reddy, M. V., and Gupta, R. C. (1981). 32P-postlabeling test for DNA damage. Proc. Natl Acad. Sci. USA 78:6126–6129.PubMedCrossRefGoogle Scholar
  13. Reddy, M. V., Bleicher, W. T., and Blackburn, G. R. (1991). 32P-postlabeling detection of thymine glycols: Evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase. Cancer Commun. 3:109–117.PubMedGoogle Scholar
  14. Stuart, G. R., and Chambers, R. W. (1987). Synthesis and properties of oligodeoxyribonucleotides with an AP site at a preselected position. Nucleic Acids Res. 15:7451–7462.PubMedCrossRefGoogle Scholar
  15. Urata, H., and Akagi, M. (1993). A convenient synthesis of oligonucleotides with a 3′-phosphoglycolate and 3′-phosphoglycoaldehyde terminus. Tetrahedron Lett. 34:4015–4018.CrossRefGoogle Scholar
  16. Weinfeld, M., and Buchko, G. W. (1993). Postlabelling methods for the detection of apurinic sites and radiation-induced DNA damage, in:Postlabelling Methods for the Detection of DNA Damage, IARC Scientific Publications No. 124 (D. H. Phillips, M. Castegnaro, and H. Bartsch, eds.), IARC Publications, Lyon, pp. 95–103.Google Scholar
  17. Weinfeld, M., and Soderlind, K.-J. (1991). 32P-postlabeling detection of radiation-induced DNA damage: Identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry 30:1091–1097.PubMedCrossRefGoogle Scholar
  18. Weinfeld, M., Liuzzi, M., and Paterson, M. C. (1989a). Enzymatic analysis of isomeric trithymidylates containing UV light-induced cyclobutane pyrimidine dimers: II. Phosphorylation by phage T4 polynucleotide kinase. J. Biol. Chem. 264:6364–6370.PubMedGoogle Scholar
  19. Weinfeld, M., Liuzzi, M., and Paterson, M. C. (1989b). Selective hydrolysis by exo-and endonucleases of phosphodiester bonds adjacent to an apurinic site. Nucleic Acids Res. 17:3735–3745.PubMedCrossRefGoogle Scholar
  20. Weinfeld, M., Liuzzi, M., and Paterson, M. C. (1990). Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: Design of a 32P-postlabeling assay for apurinic sites in DNA. Biochemistry 29:1737–1743.PubMedCrossRefGoogle Scholar
  21. Weinfeld, M., Soderlind, K.-J., and Buchko, G. W. (1993). Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA. Nucleic Acids Res. 21:621–626.PubMedCrossRefGoogle Scholar
  22. Winters, T. A., Weinfeld, M., and Jorgensen, T. J. (1992). Human HeLa cell enzymes that remove phosphoglycolate 3′-end groups from DNA. Nucleic Acids Res. 20:2573–2580.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Michael Weinfeld
    • 1
  • Michel Liuzzi
    • 2
  • George D. D. Jones
    • 3
  1. 1.Department of RadiobiologyCross Cancer InstituteEdmontonCanada
  2. 2.Bio-Méga/Boehringer Ingelheim Research Inc.LavalCanada
  3. 3.Centre for Mechanisms of Human ToxicityUniversity of LeicesterLeicesterEngland

Personalised recommendations