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

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.