Photoaffinity labeling of proteins in bovine testis nuclear extract

Biochem Biophys Res Commun. 2002 Oct 4;297(4):714-21. doi: 10.1016/s0006-291x(02)02338-0.


A binary system of photoaffinity reagents for selective affinity labeling of DNA polymerases has been developed. The photoreactive probe was formed in nuclear extract, using an end-labeled oligonucleotide containing a synthetic abasic site. This site was incised by apurinic/apyrimidinic endonuclease and then dNMPs carrying a photoreactive adduct were added to the 3(') hydroxyl using base-substituted arylazido derivatives of dUTP or dCTP. This results in the synthesis of photoreactive base excision repair (BER) intermediates. The photoreactive group was then activated, either directly (UV light exposure 320nm) or in the presence of the sensitizer of dTTP analog containing a pyrene group (Pyr-dUTP) under UV light 365nm. DNA polymerase beta was the main target crosslinked by photoreactive BER intermediates in this nuclear extract. In contrast, several proteins were labeled under the conditions of direct activation of arylazido group.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels
  • Animals
  • Cattle
  • Cell Nucleus / genetics*
  • Cell Nucleus / metabolism
  • DNA Replication
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyribonucleotides / metabolism
  • Male
  • Nuclear Proteins / genetics
  • Nuclear Proteins / isolation & purification
  • Nuclear Proteins / metabolism*
  • Testis / metabolism*
  • Tissue Extracts / metabolism


  • Affinity Labels
  • Deoxyribonucleotides
  • Nuclear Proteins
  • Tissue Extracts
  • DNA-Directed DNA Polymerase