Structural Biochemistry of Coordinated DNA Damage Repair Pathways

点击翻译按钮获取中文摘要

The long-term goal of our research program is to advance our fundamental understanding of the molecular mechanisms that underlie the coordinated action of macromolecules in biological signaling and processing pathways. Currently our focus is on the molecular underpinnings of macromolecules that orchestrate detection and repair of cellular DNA damage. All domains of life require DNA damage surveillance and repair mechanisms to preserve the integrity of the genome and thus the fitness of an organism and future generations. Our laboratory is investigating the proteins that carry out DNA damage repair, and we are currently studying the ligation repair of DNA breaks, a common final step in all pathways of DNA damage repair. We have chosen to primarily use the proteins of the thermophilic archaeon Sulfolobus Solfataricus as a model system since it preserves the essential features of eukaryotic repair systems, and the stability and compact domain structures of the proteins makes them well suited for structural analysis. Like in many organisms, S. Solfataricus DNA ligase activity is coordinated by a DNA sliding clamp protein called PCNA (proliferating cell nuclear antigen); however, the molecular basis for the coordinated action of ligase and PCNA is poorly understood. Our research program will work toward a molecular level understanding of the ligase-PCNA interaction through the use of structural biology techniques and biochemical analysis. In particular, we will investigate the conformational changes associated with the three-step mechanism of DNA ligation, and how PCNA coordinates these conformational changes. DNA damage repair requires the coordinated action of multiple enzymes, and our studies will provide key insights into how the activity of DNA ligase is coordinated by the master regulator PCNA. Notably, S. Solfataricus PCNA binds multiple components of the repair machinery (polymerase, nuclease, ligase) and thus will allow us over time to expand our analysis to the coordinated action of multiple enzymes in a complex repair pathway. The proposed research is essential to advance our fundamental understanding of the mechanisms underlying the cellular repair of DNA damage. These findings ultimately can contribute to the development of methods to disrupt efficient repair, for example in cancer cells that must cope with an inherently unstable genome and thus depend on efficient repair mechanisms.**