| Ataxia-telangiectasia, A-T, is an autosomal recessive disease caused by lack of functional ATM protein. A-T patients have cerebellar ataxia, ocular telangiectasia, immunodeficiency, radiosensitivity, cancer predisposition, elevated alphafetoprotein levels, and chromosomal instability. ATM, a protein kinase that participates in initiating repair of DNA double strand breaks, triggers signaling pathways involved in cell cycle checkpoint function, DNA repair mechanisms, and apoptosis. Phosphorylation targets of ATM include, but are not limited to, p53, MDM2, NBS1, Chk2, and BRCA1.; ATM structural studies are very limited due to lack of purified protein. Structure prediction programs and biochemical experiments have defined only a small portion of the protein to the catalytic domain and protein binding regions; 80% of the protein sequence is unknown. Problems in ATM purification are due to inherently low protein levels and expression of ATM is difficult due to the large and unstable ATM coding sequence. We describe construction of a vaccinia virus expressing FLAG-tagged ATM, expression of functional protein, and purification of at least 30 ug of ATM from 25 × 106 infected HeLa cells, to date, the highest yield reported.; We examine the kinase activity of purified FLAG-ATM using PHAS-1 and GST-p53. FLAG-ATM exhibited manganese-dependent activity that was DNA-stimulated in p53 kinase reactions, and DNA-independent in PHAS-1 reactions. ATM binding to DNA ends was seen using atomic force microscopy. Analysis using cryo-electron microscopy suggested FLAG-ATM to be spherical.; A clinical application of the purified FLAG-ATM is the ATM ELISA, a diagnostic test that can semi-quantitate ATM levels in patients. We report that the ATM ELISA could detect protein level differences between normal and A-T individuals in nuclear and whole cell lysates of lymphoblastoid cell lines (LCLs) and PBMCs.; As a final discussion, we examine radiosensitivity and ATM protein levels in A-T patients. Of 71 radiosensitive LCLs, we identified 17 LCLs that were radiosensitive but expressed normal or almost normal levels of functional nuclear ATM. Based on radiosensitivity alone, these LCLs may be misdiagnosed as A-T. We showed that typically, A-T patients have no or low amounts of ATM protein, and when protein levels were combined with radiation sensitivity status, A-T diagnosis was improved. |
