Mechanisms of Human DNA Double-Strand Break Repair via Quantitative Single-Molecule Imaging

Project summary Genetic instability and impaired DNA replication and repair are key contributors to the development of cancer and severe human syndromes. This research program focuses on studies of the mechanisms of replication-fork (RF) stability and DNA double-strand break (DSB) repair, two crucial aspects of genomic instability that can lead to cancer, and associate with therapeutic response and other human disease. DSBs are the most dangerous form of DNA damage, occurring due to normal cellular processes, exposure to ionizing radiation (IR), and chemotherapy drugs. In mammalian cells, there are two primary pathways for repairing DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR), both of which play a vital role in maintaining genomic stability. Despite the emergence of new and advanced cancer treatments, chemotherapy remains the standard- of-care, yet it generally fails to address differential response and mechanisms of resistance that often occurs. Patient-specific mutations in NHEJ and HR proteins are still the subject of intense study, and related factors are being actively pursued as potential therapeutic targets. However, the specialized steps and processes affected by these mutations remain unclear due to the limitations of standard biochemical and cellular methods in measuring critical intermediates and functional features of these repair processes. We have limited understanding regarding the toxicity of replication-fork lesions, the physical nature of molecular complexes during DSB and fork repair, their spatial and temporal organization in the cell, and their regulation and interactions at the chromatin level. Our research program aims to address these unknowns by defining key mechanistic steps of human DNA repair using an array of single-molecule assays and methods that are accompanied by contemporary cellular, biochemical and molecular biology approaches and tools. Combined, our studies will address critical unanswered questions having enormous potential for advancing the field of DNA damage research.