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J Biol Chem
2009 Nov 27;28448:33107-14. doi: 10.1074/jbc.M109.064485.
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Reconstitution of human claspin-mediated phosphorylation of Chk1 by the ATR (ataxia telangiectasia-mutated and rad3-related) checkpoint kinase.
Lindsey-Boltz LA
,
Serçin O
,
Choi JH
,
Sancar A
.
???displayArticle.abstract??? 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.
Bennett,
Regulation of Claspin degradation by the ubiquitin-proteosome pathway during the cell cycle and in response to ATR-dependent checkpoint activation.
2006, Pubmed
Bennett,
Regulation of Claspin degradation by the ubiquitin-proteosome pathway during the cell cycle and in response to ATR-dependent checkpoint activation.
2006,
Pubmed
Bennett,
Claspin is phosphorylated in the Chk1-binding domain by a kinase distinct from Chk1.
2008,
Pubmed
Chen,
Reconstitution of Rad53 activation by Mec1 through adaptor protein Mrc1.
2009,
Pubmed
Chini,
Repeated phosphopeptide motifs in human Claspin are phosphorylated by Chk1 and mediate Claspin function.
2006,
Pubmed
,
Xenbase
Chini,
Human claspin is required for replication checkpoint control.
2003,
Pubmed
,
Xenbase
Choi,
Reconstitution of a human ATR-mediated checkpoint response to damaged DNA.
2007,
Pubmed
Choi,
The human ATR-mediated DNA damage checkpoint in a reconstituted system.
2009,
Pubmed
Choi,
Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA.
2009,
Pubmed
Clarke,
DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system.
2005,
Pubmed
,
Xenbase
Finn,
The Pfam protein families database.
2008,
Pubmed
Hashimoto,
The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1.
2006,
Pubmed
,
Xenbase
Jeong,
Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation.
2003,
Pubmed
,
Xenbase
Kumagai,
TopBP1 activates the ATR-ATRIP complex.
2006,
Pubmed
,
Xenbase
Kumagai,
Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1.
2004,
Pubmed
,
Xenbase
Kumagai,
Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1.
2003,
Pubmed
,
Xenbase
Kumagai,
Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.
2000,
Pubmed
,
Xenbase
Lin,
Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation.
2004,
Pubmed
,
Xenbase
Liu,
Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.
2000,
Pubmed
Liu,
Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation.
2006,
Pubmed
Majka,
The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint.
2006,
Pubmed
Mordes,
Dpb11 activates the Mec1-Ddc2 complex.
2008,
Pubmed
Navadgi-Patil,
Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase.
2008,
Pubmed
Sancar,
Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.
2004,
Pubmed
Sar,
Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures.
2004,
Pubmed
,
Xenbase
Shiotani,
Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks.
2009,
Pubmed
Sweeney,
Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation.
2005,
Pubmed
Yan,
Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.
2006,
Pubmed
,
Xenbase
Yoo,
Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures.
2006,
Pubmed
,
Xenbase
Yoshioka,
ATR kinase activation mediated by MutSalpha and MutLalpha in response to cytotoxic O6-methylguanine adducts.
2006,
Pubmed
Zhao,
ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1.
2001,
Pubmed
