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

• Published in: FEBS letters 1999-04, Vol.448 (1), p.141-144
• Main Authors: Kolpashchikov, D.M., Rechkunova, N.I., Dobrikov, M.I., Khodyreva, S.N., Lebedeva, N.A., Lavrik, O.I.
• Format: Article
• Language: English
• Subjects: Animals; Deoxyuracil Nucleotides - metabolism; DNA Polymerase beta - metabolism; DNA-Binding Proteins - metabolism; dNTP analog; FAB-4-dUTP, 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2′-deoxyuridine-5′-triphosphate; Humans; Mammals; Molecular Structure; Peptide Chain Elongation, Translational; Photoaffinity labeling; Photoaffinity Labels - metabolism; Photosensitization; Photosensitizing Agents - metabolism; pol β, mammalian DNA polymerase β; PyrdUTP, 5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2′-deoxyuridine-5′-triphosphate; Replication Protein A; RPA, human replication protein A; Substrate Specificity
• ISSN: 0014-5793; 1873-3468
• 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 β (pol β). 5′-[ 32P]-labeled primer was elongated in situ by pol β 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 β as a result of fluorescence resonance energy transfer. The initial rate of pol β photomodification was shown to increase by a factor of ten. The selectivity of pol β photosensitized modification was proved by adding human replication protein A.