A conserved proliferating cell nuclear antigen-interacting protein sequence in Chk1 is required for checkpoint function.
Human checkpoint kinase 1 (Chk1) is an essential kinase required for cell cycle checkpoints and for coordination of DNA synthesis. To gain insight into the mechanisms by which Chk1 carries out these functions, we used mass spectrometry to identify previously uncharacterized interacting partners of Chk1. We describe a novel interaction between Chk1 and proliferating cell nuclear antigen (PCNA), an essential component of the replication machinery. Binding between Chk1 and PCNA was reduced in the presence of hydroxyurea, suggesting that the interaction is regulated by replication stress. A highly conserved PCNA-interacting protein (PIP) box motif was identified in Chk1. The intact PIP box is required for efficient DNA damage-induced phosphorylation and release of activated Chk1 from chromatin. We find that the PIP box of Chk1 is crucial for Chk1-mediated S-M and G(2)-M checkpoint responses. In addition, we show that mutations in the PIP box of Chk1 lead to decreased rates of replication fork progression and increased aberrant replication. These findings suggest an additional mechanism by which essential components of the DNA replication machinery interact with the replication checkpoint apparatus.
Scorah
Jennifer
J
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Dong
Meng-Qiu
MQ
Yates
John R
JR
Scott
Mary
M
Gillespie
David
D
McGowan
Clare H
CH
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000737:chemistry; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D006918:Hydroxyurea