Recombinant ATM protein complements the cellular A-T phenotype (original) (raw)

Oncogene volume 15, pages 159–167 (1997)Cite this article

Abstract

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability and radiation sensitivity. The cellular phenotype of A-T points to defects in signal transduction pathways involved in activation of cell cycle checkpoints by free radical damage, and other pathways that mediate the transmission of specific mitogenic stimuli. The product of the responsible gene, ATM, belongs to a family of large proteins that contribute to maintaining genome stability and cell cycle progression in various organisms. A recombinant vector that stably expresses a full-length ATM protein is a valuable tool for its functional analysis. We constructed and cloned a recombinant, full-length open reading frame of ATM using a combination of vectors and hosts that overcame an inherent instability of this sequence. Recombinant ATM was stably expressed in insect cells using a baculovirus vector, albeit at a low level, and in human A-T cells using an episomal expression vector. An amino-terminal FLAG epitope added to the protein allowed highly specific detection of the recombinant molecule by immunoblotting, immunoprecipitation and immunostaining, and its isolation using immunoaffinity. Similar to endogenous ATM, the recombinant protein is located mainly in the nucleus, with low levels in the cytoplasm. Ectopic expression of ATM in A-T cells restored normal sensitivity to ionizing radiation and the radiomimetic drug neocarzinostatin, and a normal pattern of post-irradiation DNA synthesis, which represents an S-phase checkpoint. These observations indicate that the recombinant, epitope-tagged protein is functional. Introduction into this molecule of a known A-T missense mutation, Glu2904Gly, resulted in apparent instability of the protein and inability to complement the A-T phenotype. These findings indicate that the physiological defects characteristic of A-T cells result from the absence of the ATM protein, and that this deficiency can be corrected by ectopic expression of this protein.

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Authors and Affiliations

  1. Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978, Israel
    Yael Ziv, Anat Bar-Shira, Iris Pecker & Yosef Shiloh
  2. Department of Radiation Medicine, Vincent T Lombardi Cancer Research Center, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, 20007-2197, DC, USA
    Pamela Russell & Timothy J Jorgensen
  3. Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, 69978, Israel
    Ilan Tsarfati
  4. InSight Strategy and Marketing Ltd., P.O.B. 2128, Rehovet, 76121, Israel
    Iris Pecker

Authors

  1. Yael Ziv
  2. Anat Bar-Shira
  3. Iris Pecker
  4. Pamela Russell
  5. Timothy J Jorgensen
  6. Ilan Tsarfati
  7. Yosef Shiloh

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Ziv, Y., Bar-Shira, A., Pecker, I. et al. Recombinant ATM protein complements the cellular A-T phenotype.Oncogene 15, 159–167 (1997). https://doi.org/10.1038/sj.onc.1201319

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