Abstract
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.
MeSH terms
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Acid Anhydride Hydrolases
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Animals
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Ataxia Telangiectasia Mutated Proteins
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Cell Cycle Proteins* / genetics
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Cell Cycle Proteins* / isolation & purification
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Cell Cycle Proteins* / metabolism
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DNA Damage
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DNA Repair Enzymes* / genetics
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DNA Repair Enzymes* / isolation & purification
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DNA Repair Enzymes* / metabolism
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DNA-Binding Proteins* / genetics
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DNA-Binding Proteins* / isolation & purification
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DNA-Binding Proteins* / metabolism
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Humans
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MRE11 Homologue Protein
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Multiprotein Complexes
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Nuclear Proteins* / genetics
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Nuclear Proteins* / isolation & purification
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Nuclear Proteins* / metabolism
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Protein Serine-Threonine Kinases* / genetics
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Protein Serine-Threonine Kinases* / isolation & purification
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Protein Serine-Threonine Kinases* / metabolism
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Tumor Suppressor Proteins* / genetics
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Tumor Suppressor Proteins* / isolation & purification
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Tumor Suppressor Proteins* / metabolism
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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MRE11 protein, human
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Multiprotein Complexes
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NBN protein, human
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Nuclear Proteins
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Tumor Suppressor Proteins
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ATM protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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MRE11 Homologue Protein
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Acid Anhydride Hydrolases
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RAD50 protein, human
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DNA Repair Enzymes