Single-molecule Analysis of the DNA Damage Response in Living Cells - Project Abstract DNA stores the heritable genetic information in human cells; however, DNA is constantly exposed to endogenous and exogenous insults that can cause damage, threatening genomic integrity. To counteract this damage, human cells have evolved a sophisticated machinery to detect DNA damage, the DNA damage response. The DNA damage response facilitates engagement of several pathways that can repair DNA abnormalities ranging from simple base-mismatches to highly cytotoxic double-strand breaks. Defects in either DNA repair or DNA damage response proteins underlie a wide range of human diseases, including neurological disorders, immunodeficiency, infertility, and cancer. Therefore, understanding the molecular mechanisms of DNA damage repair is highly significant for human health and could lead to new therapeutic strategies to treat these diseases. DNA repair is carried out by a variety of enzymes, including nucleases, ligases, recombinases, and other DNA modifying enzymes. Since these enzymatic activities have evolved to alter their DNA substrate, they are inherently mutagenic. It is therefore imperative that the activation of DNA repair proteins is tightly controlled and locally restricted to the site of the DNA lesion. The goal of this project is to develop and implement live cell single- molecule approaches to analyze how the DNA damage response senses genome alterations and activates the appropriate pathway to repair the lesion. We will use these methods to build a comprehensive, quantitative, mechanistic model of DNA repair in human cells. Importantly, a quantitative understanding of DNA damage response could lead to new therapeutic approaches to specifically reinforce or interfere with DNA repair in a wide range of human diseases.
