CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells.
CDC6, a replication licensing protein, is partially exported to the cytoplasm in human cells through phosphorylation by Cdk during S phase, but a significant proportion remains in the nucleus. We report here that human CDC6 physically interacts with ATR, a crucial checkpoint kinase, in a manner that is stimulated by phosphorylation by Cdk. CDC6 silencing by siRNAs affected ATR-dependent inhibition of mitotic entry elicited by modest replication stress. Whereas a Cdk-phosphorylation-mimicking CDC6 mutant could rescue the checkpoint defect by CDC6 silencing, a phosphorylation-deficient mutant could not. Furthermore, we found that the CDC6-ATR interaction is conserved in Xenopus. We show that the presence of Xenopus CDC6 during S phase is essential for Xenopus ATR to bind to chromatin in response to replication inhibition. In addition, when human CDC6 amino acid fragment 180-220, which can bind to both human and Xenopus ATR, was added to Xenopus egg extracts after assembly of the pre-replication complex, Xenopus Chk1 phosphorylation was significantly reduced without lowering replication, probably through a sequestration of CDC6-mediated ATR-chromatin interaction. Thus, CDC6 might regulate replication-checkpoint activation through the interaction with ATR in higher eukaryotic cells.
Yoshida
Kazumasa
K
Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan.
Sugimoto
Nozomi
N
Iwahori
Satoko
S
Yugawa
Takashi
T
Narisawa-Saito
Mako
M
Kiyono
Tohru
T
Fujita
Masatoshi
M
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002457:Cell Extracts; D000071877:Checkpoint Kinase 1; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication; D004789:Enzyme Activation; D005057:Eukaryotic Cells / Q000201:enzymology* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D010063:Ovum / Q000166:cytology; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D013312:Stress, Physiological; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins / Q000378:metabolism*
C103706:CDC6 protein, human; C118388:Cdc6 protein, Xenopus; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D029867:Xenopus Proteins; D011494:Protein Kinases; C576898:Atr protein, Xenopus; 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; D018844:Cyclin-Dependent Kinases