Reconstitution of human claspin-mediated phosphorylation of Chk1 by the ATR (ataxia telangiectasia-mutated and rad3-related) checkpoint kinase.
ATR (ATM and Rad3-related) initiates a DNA damage signaling pathway in human cells upon DNA damage induced by UV and UV-mimetic agents and in response to inhibition of DNA replication. Genetic data with human cells and in vitro data with Xenopus egg extracts have led to the conclusion that the kinase activity of ATR toward the signal-transducing kinase Chk1 depends on the mediator protein Claspin. Here we have reconstituted a Claspin-mediated checkpoint system with purified human proteins. We find that the ATR-dependent phosphorylation of Chk1, but not p53, is strongly stimulated by Claspin. Similarly, DNA containing bulky base adducts stimulates ATR kinase activity, and Claspin acts synergistically with damaged DNA to increase phosphorylation of Chk1 by ATR. Mutations in putative phosphorylation sites in the Chk1-binding domain of Claspin abolish its ability to mediate ATR phosphorylation of Chk1. We also find that a fragment of Claspin containing the Chk1-binding domain together with a domain conserved in the yeast Mrc1 orthologs of Claspin is sufficient for its mediator activity. This in vitro system recapitulates essential components of the genetically defined ATR-signaling pathway.
Lindsey-Boltz
Laura A
LA
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260, USA.
Serçin
Ozdemirhan
O
Choi
Jun-Hyuk
JH
Sancar
Aziz
A
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004260:DNA Repair; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins
D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D018797:Cell Cycle Proteins; D016159:Tumor Suppressor Protein p53; D029867:Xenopus Proteins; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases