RECOMBINANT ATM PROTEIN COMPLEMENTS THE CELLULAR A-T PHENOTYPE

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder charact erized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability and radiation sensitivity, The cellular phenotype o f A-T points to defects in signal transduction pathways involved in ac tivation of cell cycle checkpoints by free radical damage, and other p athways that mediate the transmission of specific mitogenic stimuli, T he product of the responsible gene, ATM, belongs to a family of large proteins that contribute to maintaining genome stability and cell cycl e progression in various organisms, A recombinant vector that stably e xpresses a full-length ATM protein is a valuable tool for its function al analysis, We constructed and cloned a recombinant, full-length open reading frame of ATM using a combination of vectors and hosts that ov ercame an inherent instability of this sequence, Recombinant ATM was s tably expressed in insect cells using a baculovirus vector, albeit at a low level, and in human A-T cells using an episomal expression vecto r, An amino-terminal FLAG epitope added to the protein allowed highly specific detection of the recombinant molecule by immunoblotting, immu noprecipitation and immunostaining, and its isolation using immunoaffi nity, Similar to endogenous ATM, the recombinant protein is located ma inly in the nucleus, with low levels in the cytoplasm, Ectopic express ion of ATM in A-T cells restored normal sensitivity to ionizing radiat ion and the radiomimetic drug neocarzinostatin, and a normal pattern o f post-irradiation DNA synthesis, which represents an S-phase checkpoi nt. These observations indicate that the recombinant, epitope-tagged p rotein is functional, Introduction into this molecule of a known A-T m issense 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 re sult from the absence of the ATM protein, and that this deficiency can be corrected by ectopic expression of this protein.