PROJECT ABSTRACT The overall goal of this proposal is to decipher a novel Ca2+-dependent signaling pathway that protects the genome in human cells in the presence of DNA replication stress. Replication stress, which can cause DNA damage and chromosomal instability, is frequently induced by both environmental agents and endogenous factors (e.g., reactive oxygen species, aldehyde, and oncogene activation). In order to maintain genome stability cells must protect the replication fork structure upon replication stress to avoid fork collapse and DNA damage. Defects in fork protection can result in cell death or transformation, which can give rise to cancer, premature ageing and other diseases. The fork protection mechanisms can also be exploited to sensitize cancer cells to replication stress-inducing agents during cancer treatment. Despite its critical importance, exactly how replication forks are protected after replication stress remain an outstanding question. In our effort to address this fundamental question, we recently discovered a novel Ca2+-dependent signaling pathway that protects stressed replication fork structure. In addition, we have established multiple components in the pathway, including CaMKK2, AMPK and Exo1, that act downstream of intracellular Ca2+, which is elevated after replication stress. Disruption of the pathway causes excessive fork degradation, chromosomal aberrations and compromised cell viability. Building on this exciting finding and our extensive preliminary results, in this proposal we describe a series of studies to further delineate this novel fork protection pathway, focusing on the key players and molecular mechanisms that mediate Ca2+ induction and pathway activation. In Aim 1, we will define a key ion channel responsible for Ca2+ induction in the replication stress response. Our preliminary results suggest that TRPV2 is a major ion channel for Ca2+ induction upon replication stress. We will characterize the molecular functions of TRPV2 in this fork protection pathway and its regulation by a novel interacting protein in the replication stress response. Aim 2 seeks to define the direct signal for Ca2+ induction after replication stress. Our preliminary results suggest that cytosolic self-DNA generated after replication stress has a previously unrecognized role in triggering Ca2+ induction for fork protection. We plan to explore the sources of cytosolic self-DNA in cells after replication stress. In addition, we will determine whether other cytosolic DNA-inducing genetic conditions also activate the Ca2+-dependent signaling cascade. In Aim 3, we will elucidate the molecular mechanisms of TRPV2 activation for Ca2+ induction after replication stress, focusing on the role of cytosolic DNA sensing and signal transduction. These studies will further establish a novel fork protection signaling pathway and provide critical insights into the interplay between replication stress, cytosolic DNA sensing and Ca2+ signaling. T...