Reconstitution of a human ATR-mediated checkpoint response to damaged DNA.
The DNA damage checkpoint response delays cell cycle progression upon DNA damage and prevents genomic instability. Genetic analysis has identified sensor, mediator, signal transducer, and effector components of this global signal transduction pathway. Here we describe an in vitro system with purified human checkpoint proteins that recapitulates key elements of the DNA damage checkpoint. We show that the damage sensor ATR in the presence of topoisomerase II binding protein 1 (TopBP1) mediator/adaptor protein phosphorylates the Chk1 signal-transducing kinase in a reaction that is strongly dependent on the presence of DNA containing bulky base lesions. The dependence on damaged DNA requires DNA binding by TopBP1, and, indeed, TopBP1 shows preferential binding to damaged DNA. This in vitro system provides a useful platform for mechanistic studies of the human DNA damage checkpoint response.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260, USA.
Jun-Hyuk
JH
Choi
Laura A
LA
Lindsey-Boltz
Aziz
A
Sancar
D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D004247:DNA; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases