Single Stranded DNA Recognition in Telomeres

描述（由申请人提供）：线性染色体终止于称为端粒的特殊核蛋白结构。这些自然末端是基因组稳定和细胞增殖不可或缺的一部分，它们的失调与癌症、衰老和其他人类疾病有关。端粒DNA异常富含gt，并以32条单链悬垂结尾，这需要一个特殊的封盖机制来防止DNA损伤机制的不适当识别。此外，端粒不能被典型的DNA复制机制完全复制，导致端粒序列在每个细胞周期中受到侵蚀。高度增殖的细胞通过复制酶端粒酶的作用克服了这种限制。本研究计划针对端粒维持的两个突出问题。第一，端粒酶全酶的成分是如何参与端粒酶的作用的？第二，端粒封盖因子如何保护染色体末端不被检测为受损DNA？生化，结构和遗传策略相结合，以了解如何端粒因子执行这些活动。这个集成程序是在一个模型系统中执行的，该模型系统可以适应多个层次的特征。第一个目的是研究体内端粒酶作用所需的两种蛋白质的特定生化和结构作用，这些蛋白质不直接负责催化。我们将验证全酶的一个因子的主要作用是通过蛋白质/蛋白质和蛋白质/核酸相互作用将端粒酶带到其作用位点的假设。第二个目标围绕着端粒封盖因子的功能。我们的结构研究强烈表明，尽管没有明显的序列相似性，但这些封盖因子采用与复制蛋白A （RPA）非常相似的三级和四级结构。使用RPA模型作为激励假设，将评估假设的capping复合体在功能上取代RPA的生化能力，以及端粒特异性特征的存在。在最后的目标中，从结构和生化研究中获得的知识被应用于高度可控的重组端粒酶测定中，以了解端粒酶活性和加工性的起源。这方面的研究将会

DESCRIPTION (provided by applicant): Linear chromosomes terminate in specialized nucleoprotein structures called telomeres. These natural ends are integral to genomic stability and cellular proliferation, and their dysregulation is linked to cancer, aging and other human diseases. Telomeric DNA is unusually GT-rich, and ends in a 32 single-strand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. Furthermore, telomeres cannot be fully replicated by the canonical DNA replication machinery, leading to erosion of telomere sequence with every cell cycle. Highly proliferating cells overcome this limitation through the action of the replicative enzyme telomerase. This research program targets two outstanding questions of telomere maintenance. First, how do the components of the telomerase holoenzyme contribute to telomerase action? Second, how do the telomere capping factors shield the end of the chromosome from detection as compromised DNA? Biochemical, structural and genetic strategies are combined to understand how telomere factors perform these activities. This integrated program is performed a model system amenable to multiple levels of characterization. The first Aim investigates the specific biochemical and structural roles of two proteins required for telomerase action in vivo that are not directly responsible for catalysis. We will test the hypothesis that the primary role of one factor of the holoenzyme is to bring telomerase to its site of action through both protein/protein and protein/nucleic acid interactions. The second Aim surrounds the function of telomere capping factors. Our structural studies strongly suggest that these capping factors adopt tertiary and quartenary structures remarkably similar to those of replication protein A (RPA), despite no discernible sequence similarities. Using the RPA model as a motivating hypothesis, the putative capping complex will be evaluated for its biochemical ability to functionally replace RPA, as well as for the presence of telomere- specific features. In the final Aim, knowledge derived from the structural and biochemical studies is applied in a highly controllable reconstituted telomerase assay to understand the origins of telomerase activity and processivity. This line of research will assess the interplay between processes that promote telomerase activity and those acting to suppress it. Throughout the research program, insights derived from these studies are validated in vivo using genetic tools and analyses of a variety of telomere phenotypes. This hypothesis-driven, highly unified research program will provide novel insights into the fundamental processes that maintain a central mechanism of chromosome stability. PUBLIC HEALTH RELEVANCE: Telomeres, the ends of linear chromosomes, play key roles in cancer and aging due to their ability to discriminate natural DNA ends from damaged DNA and by compensating for the inability of the standard replication machinery to fully copy the chromosomal terminus. The length of the telomere is a marker for aging, while the ability to properly cap telomeres is associated with genomic stability. The vast majority of human cancers activate the replicative enzyme telomerase to overcome the natural brake telomeres place on cellular proliferation. As a result of these activities, proper telomere function is integral to huan health.