Highly Selective Affinity Labeling of DNA-Polymerase from Thermus thermophilus B35 by a Binary System of Photoreactive Agents

N. I. Rechkunova^1*, D. M. Kolpashchikov¹, N. A. Lebedeva², I. O. Petruseva¹, M. I. Dobrikov¹, S. Kh. Degtyarev³, and O. I. Lavrik¹

¹Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, pr. Lavrent'eva 8, Novosibirsk, 630090 Russia; fax: (3832) 333-677; E-mail: nadyarec@niboch.nsc.ru

²Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia

³Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Sciences, ul. Timakova 2, Novosibirsk, 630117 Russia; fax: (3832) 336-853

^* To whom correspondence should be addressed.

Received June 15, 1999; Revision received October 16, 1999

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The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs. The 5´-[³²P]-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5,6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP). Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP). The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis. The enzyme was protected from the photosensitized modification by dNTP. To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture. In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled. The suggested method enables highly selective affinity modification of DNA-polymerases.

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KEY WORDS: photoaffinity labeling, TTP analogs, photosensitization, thermostable DNA-polymerase

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