PROJECT SUMMARY/ABSTRACT To preserve genomic integrity and maintain homeostasis, cells in our body must effectively respond to both exogenous and endogenous sources of DNA damage. How cellular DNA damage contribute to human disease, including neurological disorders and cancer, is a fundamental area of research. For the past thirteen years, my lab has been focused on studies related to the mechanistic basis of genomic instability. The goal of this MIRA application is to address critical gaps in our understanding of genome stability pathways and how they are differentially utilized in dividing versus non-dividing cells for the proper maintenance of cellular homeostasis. By relaying external information from the cell periphery to the nucleus, cell surface receptor tyrosine kinases (RTKs) respond to growth factors via PI3-kinase (PI3K)-AKT signaling to regulate gene expression and thereby promote growth and/or survival. Similarly, DNA damage threatens genome integrity and upon detection within the nucleus elicits DNA damage response (DDR) signaling to aid in DNA repair and cell cycle checkpoints. For our research program, we will address unique mechanisms related to how key DDR factors contribute to extracellular growth factor signaling crosstalk in dividing and non-dividing cells. To establish these mechanisms, we will utilize an array of innovative experimental approaches including genome-wide sequencing, proximity ligation proteomics, super-resolution microscopy and single-molecule tracking in live cells. We will test the hypothesis that the DDR should be viewed as a broader, stress-responsive network linking nuclear and cytoplasmic effectors to maintain physiological homeostasis through intersecting with growth factor signaling pathways. How this is achieved mechanistically will be a major focus of this application. The second project involves addressing how genotoxic stress in dividing cells impacts DNA replication dynamics and to elucidate novel molecular players that regulate replication fork recovery. Based on our innovative technique called Okazaki fragment sequencing (OK-seq), we are able to directly quantify the efficiency of replication fork initiation and termination at specific sites throughout the genome. Using this technique, we will expand our analysis to understand how genotoxic insults and DNA repair deficiencies contribute to site-specific replication fork-mediated DNA breaks using genome-wide analysis. Deciphering the mechanisms that contribute to replication-associated genomic instability may provide new avenues for targeted cancer treatment.

PROJECT NARRATIVE To survive in a changing environment, all cells and organisms must be able to adapt while preserving both metabolic homeostasis and genome integrity. Whether perturbations in genome integrity is sensed by cells to regulate growth factor signaling or DNA repair is not well-understood. Using a powerful viral model system, cutting-edge microscopy, and genomics/proteomics, we will address this current knowledge gap to understand fundamental DNA damage response processes as it relates to genome integrity in different cell types.