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An express method for testing the activity of a repair enzyme, uracil-DNA-glycosylase

Abstract

A rapid and effective method for testing a repair enzyme, uracil-DNA-glycosylase, was proposed. As a substrate, a deoxyuridine-containing 5′-32P-labeled deoxyoligonucleotide covalently attached to a polystyrene support (Tenta Gel S-NH2) was used. The ammonia cleavage of the apyrimidine site formed in the enzymic reaction followed by the transition of the labeled oligonucleotide fragment from the solid phase into solution allowed the detection of the enzymic activity.

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Abbreviations

AP site:

apyrimidine/apurine site

[3H]dU-DNA:

oligonucleotide containing [3H]-deoxyuridine

UDG:

uracil-DNA-glycosylase

References

  1. Ayala, F.J. and Kiger, J.A., Jr.,Modern Genetics, Menlo Park, CA: Benjamin/Cummings, 1984. Translated under the titleSovremennaya genetika, Moscow: Mir, 1988, vol. 2, pp. 122–127.

    Google Scholar 

  2. Devchand, P.R., McGhee, J.D., and van de Sande, H.,Nucleic Acids Res., 1993, vol. 21, pp. 3437–3443.

    PubMed  Article  CAS  Google Scholar 

  3. Greger, B. and Kemper, B.,Nucleic Acids Res., 1998, vol. 26, pp. 4432–4438.

    PubMed  Article  CAS  Google Scholar 

  4. Liu, H.-S., Tzeng, H.-C., Liang, Y.-J., and Chen, C.-C.,Nucleic Acids Res., 1994, vol. 22, pp. 4016–4017.

    PubMed  Article  CAS  Google Scholar 

  5. Ball, J.K. and Curran, R.,Anal. Biochem., 1997, vol. 253, pp. 264–267.

    PubMed  Article  CAS  Google Scholar 

  6. Delort, A.-M., Duplaa, A.-M., Molko, D., and Teoule, R.,Nucleic Acids Res., 1985, vol. 13, pp. 319–335.

    PubMed  Article  CAS  Google Scholar 

  7. Vasilenko, N.L., Bulychev, N.V., Gorn, V.V., Levina, A.S., and Nevinsky, G.A.,Mol. Biol., 1994, vol. 28, pp. 679–690.

    CAS  Google Scholar 

  8. Slupphaug, G., Eftedal, I., Kavli, B., Bharati, S., Helle, N.M., Haug, T., Levine, D.W., and Krokan, H.E.,Biochemistry, 1995, vol. 34, pp. 128–138.

    PubMed  Article  CAS  Google Scholar 

  9. Kubareva, E.A., Volkov, E.M., Vinogradova, N.L., Kanevsky, I.A., Oretskaya, T.S., Kuznetsova, S.A., Brevnov, M.G., Gromova, E.S., Nevinsky, G.A., and Shabarova, Z.A.,Gene, 1995, vol. 157, pp. 167–171.

    PubMed  Article  CAS  Google Scholar 

  10. Vinogradova, N.L., Bulychev, N.V., Maksakova, G.A., Johnson, F., and Nevinsky, G.A.,Mol. Biol., 1998, vol. 32, pp. 489–499.

    CAS  Google Scholar 

  11. Stivers, J.T.,Nucleic Acids Res., 1998, vol. 26, pp. 3837–3844.

    PubMed  Article  CAS  Google Scholar 

  12. McHugh, P.J. and Knowland, J.,Nucleic Acids Res., 1995, vol. 23, pp. 1664–1670.

    PubMed  Article  CAS  Google Scholar 

  13. Shabarova, Z.A., Bogdanov, A.A., and Zolotukhin, A.S.,Khimicheskie osnovy geneticheskoi inzhenerii (Chemical Bases of Genetic Engineering), Moscow: Izd. MGU, 1994, pp. 52–60.

    Google Scholar 

  14. Webb, T.R. and Matteucci, M.D.,J. Am. Chem. Soc., 1986, vol. 108, pp. 2764–2765.

    Article  CAS  Google Scholar 

  15. Atkinson, T. and Smith, M.,Oligonucleotide Synthesis: A Practical Approach, Gait, M.J., Ed., Oxford: IRL, 1984, pp. 35–82.

    Google Scholar 

  16. Tashlitskii, V.N. and Oretskaya, T.S.,Bioorg. Khim., 1997, vol. 23, pp. 732–741.

    Google Scholar 

  17. Neu, H.D. and Heppel, L.A.,J. Biol. Chem., 1965, vol. 240, pp. 3685–3692.

    PubMed  CAS  Google Scholar 

  18. Belavin, P.A., Netesova, N.A., Reshetnikov, S.S., Ivanisenko, V.A., Eroshkin, A.M., Protopopova, E.V., Loktev, V.B., and Malygin, E.G.,Biotechnologiya, 1997, no. 3, pp. 3–9.

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Correspondence to E. A. Kubareva.

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Sud’ina, A.E., Volkov, E.M., Oretskaya, T.S. et al. An express method for testing the activity of a repair enzyme, uracil-DNA-glycosylase. Russ J Bioorg Chem 26, 398–402 (2000). https://doi.org/10.1007/BF02758667

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  • DOI: https://doi.org/10.1007/BF02758667

Key words

  • apyrimidine site
  • enzymic activity
  • repair enzyme
  • uracil-DNA-glycosylase