The human ATR-mediated DNA damage checkpoint in a reconstituted system.
DNA damage checkpoints are signal transduction pathways that coordinate the cell cycle with other cellular responses to DNA damage in order to maintain genomic integrity. The ATR kinase is responsible for initiating the DNA damage checkpoint in response to UV-damaged DNA and incompletely replicated DNA. We have recently established an in vitro system that recapitulates most but not all features of the human ATR-mediated DNA damage checkpoint response. With this system we have shown that damaged DNA can be a direct signal for ATR activation, and that TopBP1 specifically stimulates ATR in the presence of damaged DNA under physiological ionic strength conditions. This system provides a powerful tool to gain insight into the molecular mechanism of the ATR pathway. Here we describe preparation of the checkpoint components and our specific kinase assay in more detail.
Choi
Jun-Hyuk
JH
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260, United States.
Sancar
Aziz
A
Lindsey-Boltz
Laura A
LA
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D020816:Amino Acid Motifs; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008954:Models, Biological; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016622:Silver Staining; D013379:Substrate Specificity
C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C110513:TOPBP1 protein, human; D006639:Histidine; D004247:DNA; D005982:Glutathione Transferase; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases