William G. Dunphy

Cell Cycle Checkpoints and the Preservation of Genomic Integrity

Our laboratory has been generally interested in how cells duplicate themselves successfully. For our studies, we utilize both human tissue culture cells and Xenopus egg extracts. 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 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 the Cdk family--it was historically known as Cdc2. MPF also contains a B-type cyclin.

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 more recent work has involved a study of the molecular pathways that lead to the activation of ATR. We have also been interested in the targets of this kinase and the roles of these targets in checkpoint responses. For example, we found that the activation of ATR occurs through interaction with a specific activator protein called TopBP1. We also identified a novel mediator protein called Claspin that enables activated ATR to recognize and phosphorylate Chk1. We pursued a thorough characterization of this pathway in order to elucidate new players and regulatory principles.

These efforts led to the identification of a novel replication protein called Treslin that associates physically with TopBP1. We proceeded to show that Treslin, along with a binding partner called MTBP, is absolutely essential for activation the replicative helicase at replication origins throughout the genome. We are now employing molecular and genome-wide studies to elucidate how, when, and where the Treslin-MTBP complex triggers the initiation of replication in human cells. For example, we have used the CUT&RUN method to map the genome-wide distribution of Treslin-MTBP on chromatin in human cells. These studies have illuminated key aspects of how cells choose where to begin replicating their DNA.

Overall, 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.