A New Binary System for Photosensitized Labeling of DNA Polymerases in Nuclear Extract

N. A. Lebedeva^1,2*, N. I. Rechkunova¹, S. V. Dezhurov¹, S. N. Khodyreva¹, A. Favre², and O. I. Lavrik^1,2

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

²Institut Jacques Monod (CNRS, Universite Paris 6, Universite Paris 7), 75351 Paris Cedex 05, France; fax: (33) 0144-275-716; E-mail: favre@ijm.jussieu.fr

^* To whom correspondence should be addressed.

Received April 4, 2002; Revision received June 13, 2002

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A binary system of reagents was used for photosensitized labeling of proteins of bovine testis nuclear extract. A dUTP analog containing 4-azido-2,5-difluoro-3-chloropyridyl group (FAP-dUTP) was used for the first time as a component of the binary system, and a dUTP analog containing the pyrenyl group (Pyr-dUTP) was used as a photosensitizer. Photoaffinity labeling of proteins of nuclear extract was performed using the radioactively labeled DNA duplex with the photoreactive FAP group at the 3´-end of elongating DNA strand and analog of the deoxyribose phosphate residue (3-hydroxy-2-hydroxymethyltetrahydrofuran (F) 5´-phosphate) at the 5´-end of the nick. Such structure is formed by the action of nuclear extract enzymes from the initial DNA duplex containing a synthetic apurine/apyrimidine site and is a photoreactive analog of a long-patch base excision repair intermediate. UV-irradiation modified a limited number of proteins of the nuclear extract. As shown using specific antibodies, the new binary system of photoreagents increases the efficiency of DNA polymerase beta labeling.

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KEY WORDS: photoaffinity labeling, photoreactive dNTP analogs, binary system of photoaffinity reagents, bovine testis nuclear extract

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