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专门核蛋白结构。这些天然末端是基因组稳定性和细胞增殖不可或缺的，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单链突出端结束，这需要特殊的加帽机制来防止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被侵蚀。高度增殖的细胞通过复制酶端粒酶的作用克服了这一限制。 这项研究计划针对端粒维护的两个突出问题。首先，端粒酶全酶的成分如何参与端粒酶的作用？第二，端粒加帽因子如何保护染色体末端不被检测为受损DNA？生物化学，结构和遗传策略相结合，以了解端粒因子如何执行这些活动。该集成程序是在一个模型系统中执行的，该模型系统可以进行多层次的表征。 第一个目的是研究端粒酶在体内作用所需的两种蛋白质的特定生物化学和结构作用，这两种蛋白质不直接负责催化。我们将检验这样的假设，即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。第二个目标是围绕端粒加帽因子的功能。我们的结构研究强烈表明，这些加帽因子采用三级和四级结构非常相似的复制蛋白A（RPA），尽管没有明显的序列相似性。使用RPA模型作为激励假设，将评估推定的加帽复合物在功能上替代RPA的生化能力以及端粒特异性特征的存在。在最后的目的中，来自结构和生物化学研究的知识被应用于高度可控的重组端粒酶检测，以了解端粒酶活性和持续合成能力的起源。这项研究将 评估促进端粒酶活性的过程和抑制端粒酶活性的过程之间的相互作用。在整个研究计划中，使用遗传工具和对各种端粒表型的分析，在体内验证了从这些研究中得出的见解。 这个假设驱动的，高度统一的研究计划将提供新的见解的基本过程，保持染色体稳定性的中央机制。 公共卫生相关性：端粒，线性染色体的末端，在癌症和衰老中起着关键作用，因为它们能够区分天然DNA末端与受损DNA，并补偿标准复制机制无法完全复制染色体末端。端粒的长度是衰老的标志，而适当地给端粒加帽的能力与基因组稳定性相关。绝大多数人类癌症激活复制酶端粒酶，以克服端粒对细胞增殖的天然制动作用。这些活动的结果是，适当的端粒功能是健康不可或缺的。

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.