ABSTRACT Genome instability is generally deleterious to cell growth. Paradoxically, in cancer, it is associated with increased drug resistance, metastasis and immune evasion; all of which contribute to poor patient prognosis. The broader landscape of our research is to fully understand how the DNA damage response (DDR) pathway and mitotic machinery work in concert to promote faithful genome stability. A major component of the DDR pathway is Ataxia telangiectasia and Rad3 related (ATR) kinase. I previously discovered an unexpected, and novel DNA damage- independent, role for ATR kinase after mitotic entry. This newly revealed role for ATR is essential for genome stability. This unexpected role for ATR opened up many opportunities to understand cross-talk between the pathways that regulate genomic stability and is foundational to my lab. Our long-term goal is to make unique contributions by elucidating how the non-canonical roles of the proteins that make up the DDR pathway ensure genome stability. Our five-year goal is to further our understanding of the mechanisms by which ATR promotes genome stability outside of the DNA damage response pathway in both mitosis (program 1) and interphase (program 2). Our current understanding of the mitotic function of ATR is limited to our previous observations that ATR activates promotes proper Aurora B activity. This leaves a large gap of knowledge as to how ATR promotes proper chromosome segregation. We will explore the mitotic pathways that ATR regulates, focusing on putative direct substrates that we have identified (program 1). Additionally, our previous work uncovered two novel, DDR independent functions of ATR, which we aim to fully explore in this proposal (program 2). (1) ATR directly phosphorylates lamin A/C in interphase. This has prompted us to define the function of ATR on nuclear plasticity and nuclear envelope breakdown. (2) Basal ATR activity is necessary for proper centromere identity throughout interphase. We will focus on how basal ATR activity promotes proper centromere identity and function through its regulation of promyelocytic nuclear bodies. We expect that these projects will yield critical information on the role of ATR in mitosis and how it and the DNA damage response pathway promote faithful chromosome segregation independent of DNA damage and further our understanding of the mechanism that promote genome stability.