CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells

• DOI: 10.1242/jcs.058693
• Publication Date: 2010-01-05T00:27:55Z
• Authors: Kazumasa Yoshida; Nozomi Sugimoto; Satoko Iwahori; Takashi Yugawa; Mako Narisawa-Saito; Tohru Kiyono; Masatoshi Fujita
• Keywords: Cell Extracts; DNA Replication; 0301 basic medicine; Chromosomal Proteins, Non-Histone; Nuclear Proteins; Cell Cycle Proteins; Ataxia Telangiectasia Mutated Proteins; Protein Serine-Threonine Kinases; Models, Biological; Cyclin-Dependent Kinases; 3. Good health; Enzyme Activation; 03 medical and health sciences; Eukaryotic Cells; Checkpoint Kinase 1; Mutation; Animals; Humans; Phosphorylation; Protein Kinases; HeLa Cells; Ovum; Protein Binding

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.