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

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Abstract

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 β labeling.

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Authors and Affiliations

  1. Siberian Branch of the Russian Academy of Sciences, pr. Akademika Lavrent'eva 8, Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, 630090, Russia

    N. A. Lebedeva, N. I. Rechkunova, S. V. Dezhurov, S. N. Khodyreva & O. I. Lavrik

  2. Institut Jacques Monod (CNRS, Universite Paris 6, Universite Paris 7), 75351, Paris Cedex 05, France

    N. A. Lebedeva, A. Favre & O. I. Lavrik

Authors
  1. N. A. Lebedeva
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  2. N. I. Rechkunova
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  3. S. V. Dezhurov
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  4. S. N. Khodyreva
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  5. A. Favre
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  6. O. I. Lavrik
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Lebedeva, N.A., Rechkunova, N.I., Dezhurov, S.V. et al. A New Binary System for Photosensitized Labeling of DNA Polymerases in Nuclear Extract. Biochemistry (Moscow) 68, 476–481 (2003). https://doi.org/10.1023/A:1023616400794

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  • DOI: https://doi.org/10.1023/A:1023616400794

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