Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.
ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.
Choi
Jun-Hyuk
JH
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Lindsey-Boltz
Laura A
LA
Kemp
Michael
M
Mason
Aaron C
AC
Wold
Marc S
MS
Sancar
Aziz
A
D048868:Adaptor Proteins, Signal Transducing / Q000302:isolation & purification / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D045643:DNA Repair Enzymes / Q000302:isolation & purification / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism*; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051716:Replication Protein A; C110513:TOPBP1 protein, human; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D045643:DNA Repair Enzymes