Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM.
The human neurodegenerative and cancer predisposition condition ataxia-telangiectasia is characterized at the cellular level by radiosensitivity, chromosomal instability, and impaired induction of ionizing radiation-induced cell cycle checkpoint controls. Recent work has revealed that the gene defective in ataxia-telangiectasia, termed ATM, encodes an approximately 350-kDa polypeptide, ATM, that is a member of the phosphatidylinositol 3-kinase family. We show that ATM binds DNA and exploit this to purify ATM to near homogeneity. Atomic force microscopy reveals that ATM exists in two populations, with sizes consistent with monomeric and tetrameric states. Atomic force microscopy analyses also show that ATM binds preferentially to DNA ends. This property is similar to that displayed by the DNA-dependent protein kinase catalytic subunit, a phosphatidylinositol 3-kinase family member that functions in DNA damage detection in conjunction with the DNA end-binding protein Ku. Furthermore, purified ATM contains a kinase activity that phosphorylates serine-15 of p53 in a DNA-stimulated manner. These results provide a biochemical assay system for ATM, support genetic data indicating distinct roles for DNA-dependent protein kinase and ATM, and suggest how ATM may signal the presence of DNA damage to p53 and other downstream effectors.
Smith
G C
GC
Wellcome Trust, Institute of Cancer, Department of Zoology, Tennis Court Road, Cambridge CB2 1QR, United Kingdom.
Cary
R B
RB
Lakin
N D
ND
Hann
B C
BC
Teo
S H
SH
Chen
D J
DJ
Jackson
S P
SP
D001260:Ataxia Telangiectasia / Q000235:genetics*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D018625:Microscopy, Atomic Force; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases