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

FEBS Letters

Lavrik, O.I.¹  Lebedeva, N.A.¹  Dobrikov, M.I.¹  Rechkunova, N.I.¹  Khodyreva, S.N.¹  Kolpashchikov, D.M.¹ 

[1] Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, pr. Lavrentieva 8, 630090, Novosibirsk, Russia

关键词: Photoaffinity labeling;    dNTP analog;    Photosensitization;    pol β;    mammalian DNA polymerase β;    RPA;    human replication protein A;    FAB-4-dUTP;    5-[N-(2;    3;    5;    6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2′-deoxyuridine-5′-triphosphate;    PyrdUTP;    5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2′-deoxyuridine-5′-triphosphate;   

DOI  :  10.1016/S0014-5793(99)00354-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown