The ataxia-telangiectasia, mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date. (C) 2004 Elsevier Inc. All rights reserved.