Expression and Assay of Recombinant ATM (original) (raw)

Abstract

A variety of genetic disorders involve genome instability and abnormal response to DNA damaging agents. Investigation of these disorders has revealed different metabolic pathways responsible for damage repair on one hand, and for signaling the presence of the damage to cellular regulatory systems on the other hand. Ataxia-telangiectasia (A-T) is a typical example of such a disorder. This autosomal recessive disease is characterized by degeneration of the cerebellum, thymus, and gonads, immunodeficiency, premature aging, cancer predisposition, and acute sensitivity to ionizing radiation. A-T cells show hypersensitivity to ionizing radiation and radiomimetic chemicals as well as defects in various signal-transduction pathways induced by these agents, most notably the activation of cell cycle checkpoints (1–3).

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References

  1. Shiloh Y. (1997) Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. Ann. Rev. Genet. 31, 635–662.
    Article PubMed CAS Google Scholar
  2. Gatti R. A. (1998) Ataxia-telangiectasia, in The Genetic Basis of Human Cancer (Vogelstein B. and Kinzler K. W., eds), McGraw-Hill, New York, pp. 275–
    Google Scholar
  3. Rotman G. and Shiloh Y. (1998) ATM: From gene to function. Hum, Mol. Genet. 7, 1555–1563.
    Article CAS Google Scholar
  4. Hoekstra M. F. (1997) Responses to DNA damage and regulation of cell cycle checkpoints by the ATM protein kinase family. Curr. Opin. Genet. Dev. 7, 170–175.
    Article PubMed CAS Google Scholar
  5. Jeggo P. A. (1997) DNA-PK: at the cross-roads of biochemistry and genetics. Mut. Res. 384, 1–14.
    CAS Google Scholar
  6. Brunn G. J., Hudson C. C, Sekulic A., Williams J. M., Hosoi H., Houghton P. J., Lawrence J. C, and Abraham R. T. (1997) Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science 277, 99–101.
    Article PubMed CAS Google Scholar
  7. Burnett P. E., Barrow R. K., Cohen N. A., Snyder S. H., and Sabatini D. M. (1998) RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. USA 95, 1432–1437.
    Article PubMed CAS Google Scholar
  8. Banin S., Moyal L., Shieh S.-Y., Taya Y., Anderson C. W., Chessa L., Smorodinsky N. I., Prives C, Shiloh Y., and Ziv Y. (1998) DNA damage-enhanced phosphorylation of p53 by ATM. Science 281, 1674–1677.
    Article PubMed CAS Google Scholar
  9. Canman C. E., Lim D.-S., Cimprich K. A., Taya Y., Tamai K., Sakaguchi K., Appella E., Kastan M. B., and Siliciano J. (1998) ATM is a protein kinase induced by ionizing radiation which phosphory lates p53. Science 281. 1677–1679.
    Article PubMed CAS Google Scholar
  10. Levine A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88. 323–331.
    Article PubMed CAS Google Scholar
  11. Kastan M. B., Zhan Q. El-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., and Fornace Jr. A. J. (1992) A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell, 71 589–597.
    Article Google Scholar
  12. Siliciano J. D., Canman C. E., Taya Y., Sakaguchi K., Appella E., and Kastan M. B. (1997) DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev. 11, 3471–3481.
    Article PubMed CAS Google Scholar
  13. Shieh S.-Y., Ikeda M., Taya Y., and Prives C. (1997) DNA dam age-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325–334.
    Article PubMed CAS Google Scholar
  14. Ziv Y., Bar-Shira A., Pecker I., Russell P., Jorgensen T. J., Tsarfati I., and Shiloh Y. (1997) Recombinant ATM protein complements the cellular A-T phe-notype. Oncogene 15, 159–167.
    Article PubMed CAS Google Scholar
  15. Zhang N., Chen P., Khanna K. K., Scott S., Gatei M., Kozlov S., Watters D., Spring K., Yen T., and Lavin M. (1997) Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype. Proc. Natl. Acad. Sci. USA 94, 8021–8026.
    Article PubMed CAS Google Scholar
  16. Scott S. P., Zhang N., Khamma K. K., Khromykh A., Hobson K., Watters D., and Lavin M. (1998) Cloning and expression of the ataxia-telangiectasia gene in baculovirus. Biochem Biophys. Res. Common. 245, 144–148.
    Article CAS Google Scholar
  17. Shiloh Y., Bar-Shira A., Galanty Y., and Ziv Y. (1998) Cloning and expression of large mammalian cDNAs: lessons from ATM, in Genetic Engineering, Principles and Methods (Anderson C., Brown D. D., Day P., Helsinki D., and Setlow J., eds.), Plenum Press, New York, pp. 239–248.
    Google Scholar
  18. Ziv Y., Etkin S., Danieli T., Amiel A., Ravia Y., Jaspers N. G. J., and Shiloh Y. (1989) Cellular and biochemical characteristics of an immortalized ataxia-telangiectasia (group AB) cell line. Cancer Res. 49, 2495–2501.
    PubMed CAS Google Scholar
  19. Pear W. S., Nolan G. P., Scott M. L., and Baltimore D. (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl Acad. Sci. USA 90, 8392–8396.
    Article PubMed CAS Google Scholar
  20. Petersen C. and Legerski R. (1991) High-frequency transformation of human repair-deficient cell-lines by an epstein-barr virus-based cDNA expression vector. Gene 107, 279–284.
    Article Google Scholar
  21. Hopp T. P., Prickett T. S., Price V. L., Libby R. T., March C. J., Ceretti D. P., Urdal D. L., Conion P. J. (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 6, 1204–1210.
    Article CAS Google Scholar

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Authors and Affiliations

  1. Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
    Yael Ziv, Sharon Banin & Yosef Shiloh
  2. St. Jude’s Children’s Research Hospital, Memphis, TN
    Dae-Sik Lim, Christine E. Canman & Michael B. Kastan

Authors

  1. Yael Ziv
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  2. Sharon Banin
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  3. Dae-Sik Lim
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  4. Christine E. Canman
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  5. Michael B. Kastan
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  6. Yosef Shiloh
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Editor information

Editors and Affiliations

  1. ICRF Laboratories, University of Dundee, UK
    Stephen M. Keyse

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© 2000 Humana Press Inc., Totowa, NJ

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Ziv, Y., Banin, S., Lim, DS., Canman, C.E., Kastan, M.B., Shiloh, Y. (2000). Expression and Assay of Recombinant ATM. In: Walker, J.M., Keyse, S.M. (eds) Stress Response. Methods in Molecular Biology™, vol 99. Humana Press. https://doi.org/10.1385/1-59259-054-3:99

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