Characterizing protein condensates and nuclear organization in genomic integrity

About This Grant

PROJECT SUMMARY All human cells integrate distinct DNA repair pathways and tightly control chromatin architectures to prevent genomic instability. Disruption of these critical maintenance systems or defects in any one pathway result in a mutational burden with profound physiological consequences. DNA repair in humans is predominantly performed by two mutually exclusive pathways mediated by two distinct tumor suppressors – homology directed repair (HDR) by BRCA1 and non-homologous end joining (NHEJ) by 53BP1. These tumor suppressors are examples of intrinsically disordered proteins (IDPs) containing large stretches of low complexity protein sequences. 53BP1 undergoes liquid-liquid phase separation to form biomolecular condensates in vitro and at DNA lesions and my work (during the K99 phase) establishes the essential function of 53BP1 phase separation in NHEJ. In preliminary data, I show the very first evidence that BRCA1 phase separates to form biomolecular condensates and identify a putative prion-like domain for self-assembly. Nuclear organization and chromatin structure are also vital parts of maintaining genomic integrity. DNA damage response requires dynamic rearrangements and chromatin modifications to provoke rapid repair factor recruitment to lesions. Conversely, repair factors and their complexes can also modify chromatin to drive repair programs. For example, recent studies implicate 53BP1 in maintaining heterochromatin. Despite decades of study, it is only in the past several years that condensation of tumor suppressors has been identified. While I have discovered that BRCA1 phase separates, it remains unknown by what mechanism self-assembly occurs and what role it plays in DNA repair through HDR. Further, the contributions of chromatin architecture to repair pathway selection and chromatin organization involving tumor suppressor condensates have not been characterized, leaving gaps in knowledge and possible therapeutic targets. Building on the K99 phase, the goal of this work is to characterize tumor suppressor condensation and how chromatin homeostasis contributes to genomic integrity. In Aim1, my lab will characterize the mechanisms by which BRCA1 phase separates into biomolecular condensates and how these ensembles influence DNA repair and repair pathway choice (HDR vs. NHEJ). In Aim 2, my lab will use super-resolution imaging to directly visualize and define, for the first time, the dynamic activity of chromatin in response to DNA damage and how nucleosome remodeling contribute to 53BP1- and BRCA1-mediated repair programs. Collectively, this work will address fundamental gaps in knowledge regarding the role of phase separations in genome integrity and uncover new paradigms that underlie tumor suppressor activities.