The long-term goal of the proposed research is to understand how cells preserve genomic integrity. Genetic instability is one characteristic of cancer cells and explains how they accumulate multiple genetic alterations that promote tumorigenic growth. Cells with defective DNA damage and replication stress response capabilities exhibit high rates of genomic instability. Therefore, we aim to define the components of DNA damage/replication stress response pathways and determine how they work cooperatively to prevent cancer by regulating the cell cycle, promoting DNA repair or initiating apoptosis. ATM (ataxia telangiectasia mutated) and ATR (ATM and rad3-related) function at the apex of these signaling pathways. These kinases share sequence similarity and overlapping functions such as the ability to phosphorylate BRCA1 and p53 and to regulate cell cycle transitions. However, ATM is required to signal primarily in response to DNA double strand breaks while ATR responds to many forms of DNA lesions and replication stress. The mechanisms underlying this difference remain unclear. We recently identified an ATR-interacting protein (ATRIP) that is required for ATR-dependent checkpoint signaling. We believe that ATR-ATRIP is a functional signaling unit. However, how ATRIP promotes ATR signaling remains undefined. This proposal will test the hypothesis that ATRIP is a regulatory subunit and specificity determinant for the ATR kinase through the following specific aims: (1) Define the functional domains of the ATR kinase; (2) Determine how ATRIP modifies ATR function; (3) Determine how phosphorylation of ATRIP by ATR promotes checkpoint signaling. Genetic systems to study ATR and ATRIP function will form the basis of our approach to answering these aims. Completion of this research will define how cells respond to various types of genotoxic stress and provide a mechanistic understanding of ATR-ATRIP-dependent checkpoint signaling. Checkpoint signaling pathways are frequently mutated in human cancers and their loss promotes genetic instability. In addition, many cancer therapies activate these pathways. Therefore, a mechanistic understanding of checkpoint signaling pathways is essential to understand how cancer develops and how we may better diagnose and treat cancer patients.