Elucidating mechanisms underlying replication checkpoint control

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PROJECT SUMMARY The maintenance of genomic integrity after DNA damage and replication stress depends on the coordination of DNA repair and cell cycle checkpoints. The replication checkpoint pathway, which comprises two critical protein kinases, ATR and CHK1, has an essential role in this coordination. Inhibition of the replication checkpoint results in cell lethality in response to replication stress induced by oncogenes, radiation, or chemotherapeutic agents. Indeed, several replication checkpoint inhibitors are being tested in clinical trials as potential anticancer agents. Our objective in this application is to provide a detailed mechanistic understanding of replication checkpoint control, which may help us develop the best strategies for using these checkpoint inhibitors in cancer therapy. We have studied replication checkpoint control for many years. We demonstrated that the ATR-dependent replication checkpoint can be activated by a variety of DNA lesions. In addition, ATR not only activates CHK1 but also phosphorylates many other substrates, including MCM, H2AX, and others at or near stalled replication forks to initiate replication checkpoint signaling. Moreover, we have identified and studied several key proteins such as TOPBP1, ETAA1, and Claspin involved in replication checkpoint control. Many key proteins involved in DNA replication and replication checkpoint control are essential for cell survival. Thus, it remains challenging to fully understand the roles of DNA replication and replication checkpoint proteins in cell cycle progression. Toward this end, we recently took advantage of the dTAG- mediated protein degradation system and established cell lines with inducible degradation of several essential DNA replication and replication checkpoint proteins. Initial analyses of events following the depletion of these essential proteins revealed some unexpected observations, which led us to re-evaluate replication checkpoint control and the mechanisms underlying cell cycle transitions. In this project, we will determine the essential functions of these DNA replication and checkpoint proteins in S phase and cell cycle progression. We anticipate that results from these studies will provide a better understanding of replication checkpoint control, especially how DNA replication and replication checkpoint are coordinated to ensure S phase progression and cell survival.