William G. Dunphy
Regulation of the Cell Cycle and Maintenance of Genomic Integrity
Our laboratory has been generally interested in how cells proceed through the cell cycle in an orderly manner. In order to undergo division, cells must replicate their DNA during S-phase and then distribute the duplicated copies of their genomes equally to daughter cells at M-phase or mitosis. In earlier years, we focused mainly on the enzymatic network that induces the entry of cells into mitosis. A master regulatory kinase called MPF triggers mitotic entry by phosphorylating a myriad of cellular proteins. These phosphorylations lead to the hallmark events of mitosis such as chromosome condensation, nuclear envelope disassembly, and assembly of the mitotic spindle. MPF, which stands for maturation- or mitosis-promoting factor, is a heterotrimer containing a cyclin, a cyclin-dependent kinase (Cdk), and a small ancillary protein Cks protein. The kinase subunit of MPF is Cdk1, the founding member of this family--it was historically known as Cdc2. MPF also typically contains one of the B-type cyclins.
In order for MPF to induce mitosis, it is essential that prior events in the cell cycle have occurred normally. Notably, the cell must have copied all of its genomic DNA accurately during S-phase. In addition, the DNA must also be free of damage in order for the cell to begin division. If a cell has not replicated its DNA accurately or has suffered damage in the genome, various checkpoint mechanisms impose a blockade to mitotic entry. This delay allows time for the cell to repair DNA lesions. These checkpoint responses have additional physiological consequences. For example, these pathways can influence the transcriptional program of the cell, help to stabilize aberrantly stalled replication forks, and participate in the decision to engage in apoptosis in the event of very severe damage.
Checkpoint pathways consist of sensor proteins that detect problems with the DNA and effector proteins that, for example, regulate the function of cell cycle control proteins. Various mediator proteins manage interactions between sensor and effector proteins in order to control the specificity and efficiency of checkpoint pathways. In cells with incompletely replicated DNA, a master regulatory kinase known as ATR functions near the apex of the checkpoint pathway. The action of ATR ultimately leads to the activation of a downstream effector kinase known as Chk1. A distinct kinase called ATM becomes activated in cells with various forms of damaged DNA, such as DNA with double-stranded breaks (DSBs). Both ATR and ATM are members of the phosphoinositide kinase-related family of protein kinases (PIKKs).
Much of our work now involves a study of the molecular pathways that lead to the activation of ATR. We are also interested in the targets of this kinase and the roles of these targets in checkpoint responses. In recent years, we have found that the activation of ATR occurs through interaction with a specific activator protein called TopBP1. We have also identified a novel mediator protein called Claspin that enables activated ATR to recognize and phosphorylate Chk1. We are now pursuing a thorough characterization of this pathway in order to elucidate new players and regulatory principles. These studies should eventually help us understand how cells maintain the integrity of their genomes. This issue is very relevant to human health because an overarching problem with cancer cells is that such cells have suffered a catastrophic deterioration in the mechanisms that maintain genomic stability.
Kumagai, A., and Dunphy, W.G. (2000). Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol. Cell 6, 839-849.
Guo, Z., Kumagai, A., Wang, S.X., and Dunphy, W.G. (2000). Requirement for ATR in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes and Development 14, 2745-2756.
Kumagai, A., and Dunphy, W.G. (2003). Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1. Nat. Cell Biol. 5, 161-165.
Lee, J., Kumagai, A., and Dunphy, W.G. (2003). Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. Mol. Cell 11, 329-340.
Yoo, H.Y., Kumagai, A., Shevchenko, A, Shevchenko, A., and Dunphy, W.G. (2004). Adaptation of a DNA replication checkpoint response depends upon inactivation of Claspin by the Polo-like kinase. Cell 117, 575-588.
Kumagai, A., Kim, S.-M., and Dunphy, W.G. (2004). Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1. J. Biol. Chem. 279, 49599-49608.
Kumagai, A., Lee, J., Yoo, H.Y., and Dunphy, W.G. (2006). TopBP1 activates the ATR-ATRIP complex. Cell 124, 943-955.
Yoo, H.Y., Jeong, S.-Y., and Dunphy, W.G. (2006). Site-specific phosphorylation of a checkpoint mediator protein controls its responses to different DNA structures. Genes and Development 20, 772-783.
Yoo, H.Y., Kumagai, A., Shevchenko, A., Shevchenko, A., and Dunphy, W.G. (2007). Ataxia-telangiectasia mutated (ATM)-dependent activation of ATR occurs through phosphorylation of TopBP1 by ATM. J. Biol. Chem. 282, 17501-17506.
Lee, J., Kumagai, A., and Dunphy, W.G. (2007). The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR. J. Biol. Chem. 282, 28036-28044.
Yoo, H.Y., Kumagai, A., Shevchenko, A., Shevchenko, A., and Dunphy, W.G. (2009). The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM. Mol. Biol. Cell 20, 2351-2360.
Kumagai, A., Shevchenko, A., Shevchenko, A., and Dunphy, W.G. (2010). Treslin collaborates with TopBP1 in triggering the initiation of DNA replication. Cell 140, 349-359.
Lee, J., and Dunphy, W.G. (2010). Rad17 plays a central role in establishment of the interaction between TopBP1 and the Rad9-Hus1-Rad1 complex at stalled replication forks. Mol. Biol. Cell. 21, 926-935.
Kumagai, A., Shevchenko, A., Shevchenko, A., and Dunphy, W.G. (2011). Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication. J. Cell Biol. 193, 995-1007.
Lee, J., and Dunphy, W.G. (2013). The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks. Mol. Biol. Cell. 24, 1343-1353.
Guo, C., Kumagai, A., Schlacher, K., Shevchenko, A., Shevchenko, A., and Dunphy, W.G. (2015). Interaction of Chk1 with Treslin negatively regulates the initiation of chromosomal DNA replication. Mol. Cell 57, 492-505.