Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases

FEBS Lett. 1999 Apr 1;448(1):141-4. doi: 10.1016/s0014-5793(99)00354-3.


To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA Polymerase beta / metabolism*
  • DNA-Binding Proteins / metabolism
  • Deoxyuracil Nucleotides / metabolism*
  • Humans
  • Mammals
  • Molecular Structure
  • Peptide Chain Elongation, Translational
  • Photoaffinity Labels / metabolism*
  • Photosensitizing Agents / metabolism*
  • Replication Protein A
  • Substrate Specificity


  • 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
  • DNA-Binding Proteins
  • Deoxyuracil Nucleotides
  • Photoaffinity Labels
  • Photosensitizing Agents
  • RPA1 protein, human
  • Replication Protein A
  • deoxyuridine triphosphate
  • DNA Polymerase beta