Understanding the Physical Mechanism of Telomere End Capping

端粒的适当封顶对维持染色体完整性至关重要，它可以防止降解、重组、端到端融合和DNA损伤检查点的识别。端粒盖层的缺失导致灾难性的染色体损伤。虽然在体内参与这些功能的蛋白质已经被确定，但关于它们在体外的功能的信息却很少。需要生化和结构数据来开发可行的端粒功能机制模型。本研究计划的目的是获得详细的生化和结构数据，以确定端粒封顶的机制。构成酿酒葡萄球菌染色体端帽的蛋白质被遗传学定义为Cdc13、Stn1和Ten1。首席研究员将确定由这些蛋白质介导的capping活性的物理基础。将测量蛋白质/蛋白质和蛋白质/核酸相互作用，以开发端粒末端保护复合物的完整生化理解。核磁共振将用于绘制端盖配合物的分子间界面。通过在体内和体外对这些界面进行诱变，可以进一步了解末端保护的机制。此外，研究者将研究端粒酶活性的存在和不存在的封盖成分。这个假设驱动的研究项目将通过一系列重点和优先目标，为封盖复杂地层的机制提供重要的生化和结构见解。这个程序的完成将导致对真核细胞中必不可少的端粒维持过程的重要机制理解。该项目还将广泛使用多机构高场核磁共振设备，为项目参与者提供良好的培训机会，并支持科学基础设施。此外，该项目将增加在科学领域代表性不足的群体的培训机会。PI将继续为来自代表性不足群体的学生、博士后研究员和年轻教师提供指导活动，并将以少数民族的本科院校为目标进行推广。

Proper capping of telomeres is critical for maintaining chromosomal integrity by preventing degradation, recombination, end-to-end fusions and recognition by DNA damage checkpoints. The absence of telomere capping leads to catastrophic chromosomal damage. While the proteins that participate in these functions in vivo have been identified, scant information on their function in vitro is available. Biochemical and structural data are needed to develop viable mechanistic models for telomere function. The objective of this research program is to obtain detailed biochemical and structural data to determine the mechanism of telomere capping. The proteins comprising the S. cerevisiae chromosomal end cap have been genetically defined as Cdc13, Stn1 and Ten1. The principal investigator will determine the physical basis for capping activity mediated by these proteins. Protein/protein and protein/nucleic acid interactions will be measured to develop a complete biochemical understanding of the telomere end-protection complex. NMR will be used to map intermolecular interfaces of the end-capping complexes. Insights into the mechanism of end protection will be obtained by mutagenesis of these interfaces in vitro and in vivo. Furthermore, the investigator will study telomerase activity in the presence and absence of capping components. This hypothesis-driven research program will, through a series of focused and prioritized objectives, provide significant biochemical and structural insights into the mechanism of capping complex formation.Completion of this program will lead to important mechanistic understanding of telomere maintenance, an essential process in eukaryotic cells. The program will also make extensive use of multi-institutional high-field NMR facilities, providing both an excellent training opportunity for program participants as well as supporting the scientific infrastructure. In addition, the program will increase training opportunities for groups under-represented in science. The PI will continue mentoring activities for students, postdoctoral fellows and young faculty from under-represented groups, and will target undergraduate institutions with minority populations for outreach.