Purification and biochemical characterization of ataxia-telangiectasia mutated and Mre11/Rad50/Nbs1.
Ataxia-telangiectasia mutated (ATM) is a serine-threonine kinase that is activated by DNA double strand breaks to phosphorylate many cellular proteins involved in cell cycle regulation and DNA repair. We have shown previously that the activation of ATM can be reconstituted in an in vitro system using recombinant human ATM. In this system, ATM activity is dependent on the Mre11/Rad50/Nbs1 (MRN) complex and linear DNA, similar to requirements observed in human cells. This chapter describes methods used for the overexpression and purification of human ATM and MRN, as well as a protocol for in vitro kinase assays.
Lee
Ji-Hoon
JH
Institute of Cellular and Molecular Biology, University of Texas at Austin, USA.
Paull
Tanya T
TT
D017766:Acid Anhydride Hydrolases; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D000076228:MRE11 Homologue Protein; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D046912:Multiprotein Complexes; C112156:NBN protein, human; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes