Signaling Mechanism of the DNA Replication Checkpoint

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DESCRIPTION (provided by applicant): Project Summary DNA replication checkpoint is a highly conserved signaling pathway in all eukaryotes. It plays a critical role in maintaining the DNA synthesis function of perturbed replication forks under stress. Perturbed forks, if undetected by the checkpoint, undergo catastrophic collapse resulting in chromosomal DNA damage or even cell death. Defects in the pathway are linked to genome instability and cancer. However, despite its importance and intense research efforts, our understanding of the mechanisms involved in the initiation of checkpoint signaling at the pertubed forks, fork stabilization, and cell survival remains incomplete. The long-term goal of our research is to understand the molecular interactions between the replication machinery and the checkpoint pathways for proper checkpoint signaling and fork protection by using S. pombe as the model system. The objective here is to define the important checkpoint functions of DNA polymerase Pol �, the replicative helicase CMG, and the sterol synthesis enzyme Erg11 (Cyp51 in humans). Our central hypotheses are (1) that the activated replication checkpoint targets Pol � and CMG on the leading strand to suppress the fork progression under stress and hence protect the perturbed forks against catastrophic collapse, and (2) that Erg11 may function as a new sensor of the stress induced by the ribonucleotide reductase inhibitor hydroxyurea. Our hypotheses are the results of our strong preliminary data and recent publications. The rationale for the proposed research is that understanding the checkpoint functions of these essential enzymes will provide novel insights into how the replication checkpoint signaling is initiated and how perturbed forks are stabilized for cell survival, the two most prominent questions in the field. Guided by strong preliminary data, these hypotheses will be tested by pursuing three specific aims: (1) determine how Pol � is regulated by the checkpoint for stabilization of perturbed forks; (2) discover the major target() of the replication checkpoint for cell survival under stress; and (3) define the functions of Erg11 in checkpoint signaling and hydroxyurea-induced cytokinesis arrest. Under the first two aims, we will conduct in vitro and in vivo studies to investigate how the activties of Pol � and CMG are regulated by the chekcpoint for fork protection and cell survival. We will also systematically analyze all replication proteins in fission yeast in order to identify the major checkpoint target() that may work alone or redundantly with the known targets. Under the third aim, the newly identified functions of Erg11 in checkpoint signaling and cytokinesis will be characterized. The approach is innovative, because it aims to provide a comprehensive molecular mechanism for checkpoint signaling in a model system representative of higher eukaryotes. The proposal is significant, because it is expected to vertically advance and expand our understanding of how checkpoint signaling is generated at perturbed forks and how perturbed forks are protected. Ultimately, such knowledge will advance our understanding of how genomic integrity is maintained and how it can be disrupted in all eukaryotes.