An Immunoassay for Measuring Repair of UV Photoproducts

  • Shirley McCready
Part of the Methods in Molecular Biology book series (MIMB, volume 1105)


A method is described that makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UVC)—pyrimidine-pyrimidone 6-4 photoproducts ([6-4]PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irradiated cells is applied to a nitrocellulose dot-blot and quantitated using an enzyme-conjugated secondary antibody and a color assay. Although the polyclonal antiserum contains antibodies to both [6-4]PPs and CPDs, repair of these lesions can be measured separately by differential destruction or repair of one or other photoproduct. The method is useful for measuring repair in total genomic DNA, and is sufficiently sensitive to measure repair of damage induced by doses of 10 J/m2 of UVC and less. The method is very versatile and has been used to measure repair in human cells, yeasts, plants, archaea, bacteria, and filamentous fungi.

Key words

UV damage DNA repair Cyclobutane dimmers (6-4) Photoproducts Immunoassay Dot blot 


  1. 1.
    McCready SJ, Cox BS (1993) The repair of 6-4 photoproducts in Saccharomyces cerevisiae. Mutat Res 293:233–240PubMedCrossRefGoogle Scholar
  2. 2.
    McCready SJ, Carr AM, Lehmann AR (1993) The repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in Schizosaccharomyces pombe. Mol Microbiol 10:885–890PubMedCrossRefGoogle Scholar
  3. 3.
    McCready S, Marcello L (2003) Repair of UV damage in Halobacterium salinarum. Biochem Soc Trans 31:694–698PubMedCrossRefGoogle Scholar
  4. 4.
    Mitchell DL, Clarkson JM (1981) The development of a radioimmuno-assay for the detection of photoproducts in mammalian cell DNA. Biochem Biophys Acta 655:54–60PubMedGoogle Scholar
  5. 5.
    Mitchell DL, Nairn RS (1989) The biology of the (6-4) photoproduct. Photochem Photobiol 49:805–820PubMedCrossRefGoogle Scholar
  6. 6.
    Sancar A, Smith FW, Sancar GB (1984) Purification of Escherichia coli DNA photolyase. J Biol Chem 259:6028–6032PubMedGoogle Scholar
  7. 7.
    Lamola AA (1969) Specific formation of thymine dimers in DNA. Photochem Photobiol 9: 291–294PubMedCrossRefGoogle Scholar
  8. 8.
    Moore D (1988) Purification and concentration of DNA from aqueous solutions. In: Ausubel FM, Brent R, Kingston RE et al (eds) Current protocols in molecular biology, vol 1. Wiley, New York, p 2.1.1Google Scholar
  9. 9.
    Wani AA, d’Ambrosio SM, Nasir AK (1987) Quantitation of pyrimidine dimers by immunoslot blot following sublethal UV-irradiation of human cells. Photochem Photobiol 46: 477–482PubMedCrossRefGoogle Scholar
  10. 10.
    Cleaver JE, Cortes F, Lutze L, Morgan WF, Player AN, Mitchell DL (1987) Unique DNA repair properties of a xeroderma pigmentosum revertant. Mol Cell Biol 7: 3353–3357PubMedCentralPubMedGoogle Scholar

Copyright information

© Humana Press 2014

Authors and Affiliations

  1. 1.School of Biological and Molecular SciencesOxford Brookes UniversityOxfordUK

Personalised recommendations