Cdc7-dependent and -independent phosphorylation of claspin in the induction of the dna replication checkpoint
Rainey, Michael ; Harhen, Brendan ; Wang, Guan-Nan ; Murphy, Paul ; Santocanale, Corrado
Rainey, Michael
Harhen, Brendan
Wang, Guan-Nan
Murphy, Paul
Santocanale, Corrado
Repository DOI
Publication Date
2013-05-15
Type
Article
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Citation
Rainey, Michael; Harhen, Brendan; Wang, Guan-Nan; Murphy, Paul; Santocanale, Corrado (2013). Cdc7-dependent and -independent phosphorylation of claspin in the induction of the dna replication checkpoint. Cell Cycle 12 (10), 1560-1568
Abstract
Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin's function in the DNA replication checkpoint.
Funder
Publisher
Informa UK Limited
Publisher DOI
10.4161/cc.24675
Rights
Attribution-NonCommercial-NoDerivs 3.0 Ireland