Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents

Biochemistry (Mosc). 2000 Feb;65(2):244-9.


The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs. The 5;-[32P]-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5, 6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP). Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP). The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis. The enzyme was protected from the photosensitized modification by dNTP. To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture. In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled. The suggested method enables highly selective affinity modification of DNA-polymerases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels*
  • Base Sequence
  • DNA Primers / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyuracil Nucleotides / pharmacology
  • Humans
  • In Vitro Techniques
  • Photosensitizing Agents / pharmacology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Replication Protein A
  • Thermus thermophilus / enzymology*
  • Thymine Nucleotides / pharmacology


  • 5-(N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate
  • 5-(N-(4-(1-pyrenyl)butylcarbonyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate
  • Affinity Labels
  • DNA Primers
  • DNA-Binding Proteins
  • Deoxyuracil Nucleotides
  • Photosensitizing Agents
  • RPA1 protein, human
  • Recombinant Proteins
  • Replication Protein A
  • Thymine Nucleotides
  • DNA-Directed DNA Polymerase
  • thymidine 5'-triphosphate