Roles of CST, a novel telomere associated complex, at the DNA replication fork

描述（由申请人提供）：该项目的总体目标是通过DNA的难以复制的位点来了解DNA复制的机制。这些将包括预测形成DNA二级结构的位点，如端粒和脆性位点。端粒由重复的、富含G-C的DNA组成，长期以来被认为是复制叉机制的天然障碍。脆性位点表达为在复制应激条件下发生的染色体DNA中的缺口或断裂。端粒和脆性位点已知会导致基因组不稳定，并且在不适当维护时可能导致致癌作用。CTC 1-STN 1-TEN 1（CST）是最近在植物和脊椎动物中发现的一种新的蛋白质复合物。STN 1和TEN 1是酵母蛋白的同源物，它们参与端粒的维持和末端保护。在人类中，CST被证明定位于端粒。人CST亚基的消耗导致基因组不稳定性的标志，即增加的3 H2 AX焦点和染色质桥，以及端粒异常，如增加的G-突出端和端粒信号丢失。多个实验室的研究结果表明，CST具有端粒和非端粒功能.重要的是，CST被证明与DNA聚合酶1-引物酶（pol 1）相互作用，启动DNA合成。本提案中提出的初步结果表明，CST在端粒复制和复制叉重新启动中发挥作用。我们认为，CST招募DNA聚合酶1重新启动DNA合成的网站，难以复制的DNA，这可能会停止DNA复制。在目的1中，将研究CST在端粒复制中的作用。然后将扩展研究以确定是否需要CST来维持染色体脆性位点。最后，CST和RecQ解旋酶，WRN和BLM，需要维持端粒和脆性位点的稳定性之间的相互作用的程度将被测量。在目标2中，将研究CST促进复制叉进展的机制。将在CST耗尽的细胞系中测量叉停滞后的叉重新启动和单链DNA的水平。CST与复制叉组件和叉重新启动因素的相互作用，然后将进行探讨。总之，这些研究将有助于理解CST如何通过难以复制的DNA来帮助维持基因组稳定性。 公共卫生相关性：每次细胞分裂时，它的DNA必须正确地复制或复制;如果在复制过程中发生错误，可能导致突变，从而导致疾病，如癌症和遗传疾病。一些细胞DNA由于不寻常的DNA结构而固有地难以复制，并且可能需要特殊蛋白质的作用来促进适当的复制。拟议的研究将探索新发现的蛋白质复合物在这些难以复制的位点复制中的作用。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand the mechanism of DNA replication through difficult-to-replicate sites of DNA. These would include sites predicted to form DNA secondary structure such as telomeres and fragile sites. Telomeres consist of repetitive, G-C rich DNA and have long been considered natural impediments to the replication fork machinery. Fragile sites are expressed as gaps or breaks in chromosomal DNA that occur during conditions of replication stress. Telomeres and fragile sites are known to cause genomic instability and can lead to carcinogenesis when not properly maintained. CTC1-STN1-TEN1 (CST) is a novel protein complex, which was recently discovered in plants and vertebrates. STN1 and TEN1 are homologues to yeast proteins, which are involved in telomere maintenance and end-protection. In humans, CST was shown to localize to telomeres. Depletion of human CST subunits results in hallmarks of genomic instability, i.e. increased 3H2AX foci and chromatin bridges, as well as telomere abnormalities such as increased G-overhangs and telomere signal loss. Results from several labs suggest that CST has both telomeric and non- telomeric functions. Importantly, CST was shown to interact with DNA polymerase 1-primase (pol 1), which initiates DNA synthesis. Preliminary results presented in this proposal suggest that CST functions in telomere replication and replication fork reinitiation. We propose that CST recruits DNA pol 1 to reinitiate DNA synthesis at sites of dificult-to-replicate DNA, which may stall DNA replication. In aim 1, the role of CST in telomere replication will be investigated. Studies will then be extended to determine whether CST is required to maintain chromosomal fragile sites. Finally, the extent to which interactions between CST and the RecQ helicases, WRN and BLM, are required to maintain telomere and fragile site stability will be measured. In aim 2, the mechanism by which CST promotes replication fork progression will be studied. Fork reinitiation after fork stalling and the levels of single-stranded DNA will be measured in CST-depleted cell lines. CST interactions with replication fork components and fork reinitiation factors will then be explored. Together, these studies will provide an understanding of how CST helps maintain genomic stability through difficult-to-replicate DNA. PUBLIC HEALTH RELEVANCE: Each time a cell divides its DNA must be copied, or replicated, properly; if mistakes occur during replication, mutations can result which can lead to diseases, such as cancer and genetic disorders. Some cellular DNA is inherently difficult to replicate due to unusual DNA structure and may require the action of special proteins to promote proper replication. The proposed research will explore the role a newly discovered protein complex in replication of these difficult-to-replicate sites.