title: A human cell extract-based assay for the activation of ATM and ATR checkpoint kinases.
abstract: The Ataxia telangiectasia-mutated (ATM) and the ATM-Rad3-related (ATR) kinases are master regulators of the DNA damage-signaling pathways that respond to a wide variety of DNA damage. In this chapter, we describe an in vitro biochemical assay to study the activation of ATM and ATR by double-stranded DNA breaks (DSBs) (Shiotani and Zou, 2009, Mol Cell 33, 547-58). In this assay, DNA fragments with different structural features are used to activate ATM and ATR in human cell extracts, and the activation of ATM and ATR is monitored by the phosphorylation of specific ATM and ATR substrates. Importantly, in this assay both ATM and ATR are activated in a DNA structure-regulated manner, providing a useful tool to characterize the DNA structural determinants for their activation. The four primary steps of this assay are as follows: (1) preparation of nuclear extracts from cultured human cells; (2) generation of various DNA fragments using DNA oligonucleotides or plasmids; (3) incubation of DNA fragments in extracts; (4) analysis of the phosphorylation of specific ATM or ATR substrates.
authors.affiliation: Harvard Medical School, Massachusetts General Hospital Cancer Center, Charlestown, MA, USA. bshiotani@partners.org
authors.forename: Bunsyo
authors.initials: B
authors.lastname: Shiotani
authors.forename: Lee
authors.initials: L
authors.lastname: Zou
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases