Functional Analysis of Telomere Protein Complexes

端粒是真核生物染色体的末端，由双链富含G重复序列组成的长串联阵列以单链突起形式延伸到染色体的3‘端。端粒有两个基本功能：作为端粒酶复制的模板和保护染色体末端不受DNA修复系统的影响。端粒功能对染色体稳定和细胞生长至关重要，而端粒的完整性影响细胞衰老。人类端粒被结合双链DNA的蛋白质TRF1和TRF2所覆盖。直到最近，TRF1和TRF2(连同其独特的相关因子)被认为分别独立地调节端粒长度和保护染色体末端。然而，新的研究表明，TRF1和TRF2作为部分冗余的复合体存在，每个都与一组共同的端粒蛋白结合：TIN2、TPP1(TINT1/PIP1/PTOP)和POT1。这些关于端粒冗余复合体的新发现挑战了关于端粒功能如何在分子水平上调节的既定范式。这个项目的目的是确定这些部分冗余的复合体如何发挥作用(无论是分散的还是共同的)来调节端粒长度和保护染色体末端。起点将是对组件之间相互作用区域的分子解剖。TIN2是部分冗余复合体的焦点。TIN2结合TRF1、TRF2和TPP1。TPP1将该复合体与POT1连接起来。为了确定每个交互作用对端粒功能的贡献，将产生TIN2和TPP1等位基因，其中一个或多个交互作用被点突变破坏，但其他交互作用保持不变。将建立稳定表达突变等位基因的细胞系，并进行功能分析，以确定每个突变对体内端粒长度调节和/或染色体末端保护的影响。尽管这些复合体看起来是多余的，但它们可以通过与新蛋白质的结合来区分。因此，为了确定可能区分这些复合体的新蛋白质，将进行双杂交筛选和免疫共沉淀分析。最终，对单个成分、它们独特的结合伙伴及其功能的分子解剖将是理解端粒功能的关键。端粒是真核染色体末端的专门结构，对于这些染色体的稳定性是必不可少的。端粒的功能由与端粒DNA结合的多亚单位蛋白质复合体控制。在这个项目中，研究人员将阐明这些蛋白质复合体调节和保护这些基本结构的分子机制。史密斯博士认为，指导学生是大学研究中一个重要且富有成效的组成部分。她指导过的人包括女性和少数族裔，范围从高中生到研究生。她发现，她用来可视化人类细胞和染色体的显微技术尤其吸引了年轻研究人员的兴趣。在这项研究中，她纳入了一些项目，这些项目具有专门为吸引和适合这类研究人员而设计的功能。

Telomeres, the ends of eukaryotic chromosomes, consist of long tandem arrays of double-stranded G-rich repeats that run out as single-stranded overhangs to the 3'-ends of chromosomes. Telomeres have two essential functions; serving as templates for replication by the enzyme telomerase and protecting chromosome ends from DNA repair systems. Telomere function is essential for chromosome stability and cell growth, and telomere integrity impacts cellular aging. Human telomeres are coated by the proteins TRF1 and TRF2, which bind double-stranded DNA. Until recently, TRF1 and TRF2 (along with their unique associated factors) were believed to function independently to regulate telomere length and protect chromosome ends, respectively. However, new studies show that TRF1 and TRF2 exist as partially redundant complexes, each bound to a common set of telomeric proteins: TIN2, TPP1 (TINT1/PIP1/PTOP), and POT1. These new findings of redundant complexes at telomeres challenge the established paradigms on how telomere function is regulated at the molecular level. The object of this project is to determine how these partially redundant complexes function (either discretely or together) to regulate telomere length and protect chromosome ends. The starting point will be a molecular dissection of the interacting domains between the components. TIN2 is the focal point of the partially redundant complexes. TIN2 binds TRF1, TRF2, and TPP1. TPP1 links the complex to POT1. To determine the contribution of each interaction to telomere function, alleles of TIN2 and TPP1 will be generated in which one or more interactions are ablated by point mutations, but other interactions are maintained. Stable cell lines expressing the mutant alleles will be generated and functional analyses performed to determine the effect of each mutation on telomere length regulation and/or chromosome end protection in vivo. Although these complexes appear redundant they may be distinguished by association with novel proteins. Thus, to identify new proteins that might distinguish the complexes, two-hybrid screens and coimmunoprecipitation analysis will be performed. Ultimately, the molecular dissection of the individual components, their unique binding partners, and their function will be essential for understanding telomere function.Telomeres, the specialized structures at the ends of eukaryotic chromosomes, are essential for the stability of those chromosomes. Telomere function is controlled by multisubunit protein complexes that bind to the telomeric DNA. In this project the investigator will elucidate the molecular mechanisms by which these protein complexes regulate and protect these essential structures. Dr. Smith considers mentoring students an important and productive component of university research. Individuals she has mentored include women and minorities and range from high school students to graduate students. She has found that the microscopic techniques she uses to visualize human cells and chromosomes especially capture the interest of younger researchers. For this research she has incorporated projects with features specifically designed to appeal to, and be appropriate for, this category of researchers.