Highly efficient labeling of DNA polymerases by a binary system of photoaffinity reagents

Biochemistry (Mosc). 2002 Jul;67(7):807-14. doi: 10.1023/a:1016309010195.


A binary system of photoaffinity reagents was proposed earlier for highly efficient labeling of DNA polymerases by 5;-[32P]DNA primers. In the present study we demonstrate the feasibility of this approach to increase the efficiency of DNA polymerase labeling. A photoactive 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was incorporated at the 3;-end of 5;-[32P]DNA primers synthesized by DNA polymerase beta or Tte in the presence of one of the dTTP analogs--FAB-4-dUTP, FAB-9-dUTP, or FAB-4-ddUTP. The reaction mixture was irradiated by light with wavelength of 334-365 nm (direct labeling) or 365-450 nm in the presence of photosensitizer, one of dTTP analogs containing a pyrene moiety, Pyr-6-dUTP or Pyr-8-dUTP. In the case of the binary system of photoaffinity reagents, a FAB group is activated by energy transfer from sensitizer localized in the dNTP-binding site of DNA polymerase in the triple complex, comprised by reagent, DNA polymerase, and Pyr-6(8)-dUTP. Direct activation of the FAB group under these conditions is negligible. The most efficient photolabeling of DNA polymerases was observed with a primer containing a FAB-4-dUMP group at the 3;-end, and Pyr-6-dUTP as a photosensitizer. Using 10-fold molar excess of photoreagent to DNA polymerase beta, the labeling efficiency was shown to achieve 60%, which is 2-fold higher than the efficiency of the direct DNA polymerase labeling under harsher conditions (334-365 nm).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Polymerase beta / metabolism*
  • DNA Primers / chemistry
  • DNA Primers / metabolism
  • Deoxyuracil Nucleotides / chemistry
  • Deoxyuracil Nucleotides / metabolism
  • Molecular Structure
  • Photoaffinity Labels / chemistry
  • Photoaffinity Labels / metabolism*
  • Photosensitizing Agents / chemistry
  • Photosensitizing Agents / metabolism
  • Templates, Genetic


  • 5-(N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate
  • DNA Primers
  • Deoxyuracil Nucleotides
  • Photoaffinity Labels
  • Photosensitizing Agents
  • DNA Polymerase beta