ATM protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation.
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.
Department of Pathology, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
Helen H
HH
Chun
Robert B
RB
Cary
Fredrick
F
Lansigan
Julian
J
Whitelegge
David J
DJ
Rawlings
Richard A
RA
Gatti
D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites; D018797:Cell Cycle Proteins; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D025521:Tumor Suppressor Proteins; D014616:Vaccinia virus / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D004247:DNA; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases