Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.
TopBP1-like proteins, which include Xenopus laevis Xmus101, are required for DNA replication and have been linked to replication checkpoint control. A direct role for TopBP1/Mus101 in checkpoint control has been difficult to prove, however, because of the requirement for replication in generating the DNA structures that activate the checkpoint. Checkpoint activation occurs in X. laevis egg extracts upon addition of an oligonucleotide duplex (AT70). We show that AT70 bypasses the requirement for replication in checkpoint activation. We take advantage of this replication-independent checkpoint system to determine the role of Xmus101 in the checkpoint. We find that Xmus101 is essential for AT70-mediated checkpoint signaling and that it functions to promote phosphorylation of Claspin bound Chk1 by the ataxia-telangiectasia and Rad-3-related (ATR) protein kinase. We also identify a separation-of-function mutant of Xmus101. In extracts expressing this mutant, replication of sperm chromatin occurs normally; however, the checkpoint response to stalled replication forks fails. These data demonstrate that Xmus101 functions directly during signal relay from ATR to Chk1.
Yan
Shan
S
The Biological Laboratories, Harvard University, Cambridge, MA 02138, USA.
Lindsay
Howard D
HD
Michael
W Matthew
WM
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D046912:Multiprotein Complexes / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; C416368:CLSPN protein, Xenopus; D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C510726:Xmus101 protein, Xenopus; D011494:Protein Kinases; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases