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

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 L L (pol L L). 5P-[ QP P]-labeled primer was elongated in situ by pol L L with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365^450 nm), can activate the photoreagent, crosslinking it to pol L L as a result of fluorescence resonance energy transfer. The initial rate of pol L L photomodification was shown to increase by a factor of ten. The selectivity of pol L L photosensitized modification was proved by adding human replication protein A.