Structural Biochemistry of Coordinated DNA Damage Repair Pathways

我们研究计划的长期目标是推进我们对生物信号和处理途径中大分子协调作用的分子机制的基本理解。目前，我们的研究重点是协调细胞DNA损伤检测和修复的大分子的分子基础。生命的所有领域都需要DNA损伤监测和修复机制来保持基因组的完整性，从而保证生物体和后代的健康。我们的实验室正在研究进行DNA损伤修复的蛋白质，我们目前正在研究DNA断裂的结扎修复，这是所有DNA损伤修复途径中常见的最后一步。我们选择主要使用嗜热古细菌Sulfolobus Solfataricus的蛋白质作为模型系统，因为它保留了真核修复系统的基本特征，并且蛋白质的稳定性和紧凑的结构域结构使它们非常适合进行结构分析。与许多生物一样，S. Solfataricus的DNA连接酶活性由一种称为PCNA（增殖细胞核抗原）的DNA滑动夹蛋白协调；然而，连接酶和PCNA协调作用的分子基础尚不清楚。我们的研究计划将通过使用结构生物学技术和生化分析，在分子水平上了解连接酶- pcna相互作用。特别是，我们将研究与DNA连接三步机制相关的构象变化，以及PCNA如何协调这些构象变化。DNA损伤修复需要多种酶的协同作用，我们的研究将为DNA连接酶的活性如何由主调节因子PCNA协调提供关键见解。值得注意的是，S. Solfataricus的PCNA结合了修复机制的多种成分（聚合酶、核酸酶、连接酶），因此随着时间的推移，我们可以将分析扩展到复杂修复途径中多种酶的协调作用。提出的研究是必要的，以促进我们的基本机制的理解，潜在的DNA损伤的细胞修复。这些发现最终有助于开发破坏有效修复的方法，例如，必须应对固有不稳定的基因组并因此依赖于有效修复机制的癌细胞。

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.