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

CST（一种新型端粒相关复合物）在 DNA 复制叉中的作用

• 批准号: 8123664
• 负责人: Jason Aaron Stewart
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-18 至 2013-04-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8123664
• 关键词: Aphidicolin; Binding Proteins; Cell Cycle; Cell Cycle Progression; Cell Line; Cells; Chromatin; Chromosome Fragile Sites; Chromosome abnormality; Chromosomes; Complex; DNA; DNA Damage; DNA Primase; DNA Sequence; DNA Structure; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Defect; Disease; Fluorescent in Situ Hybridization; Genome; Genome Stability; Genomic Instability; Goals; Hereditary Disease; Homologous Gene; Human; Immunoprecipitation; Lead; Measures; Metaphase Spread; Mutation; Pathway interactions; Plants; Polymerase; Proteins; Recovery; Recruitment Activity; Research; Role; Signal Transduction; Single-Stranded DNA; Site; Stress; Structure; TERF1 gene; Telomere Maintenance; Testing; Time; Vertebrates; base; cancer genetics; carcinogenesis; cell type; helicase; insight; novel; overexpression; protein complex; replication factor A; research study; telomere; yeast protein

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.

描述（由申请人提供）：该项目的总体目标是通过DNA难以复制的位点了解DNA复制的机制。这些将包括预测形成DNA二级结构的位点，如端粒和脆弱位点。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器的天然障碍。脆弱位点表现为染色体DNA在复制应激条件下出现的间隙或断裂。众所周知，端粒和脆弱位点会导致基因组不稳定，如果维护不当，可能导致致癌。CTC1-STN1-TEN1 （CST）是近年来在植物和脊椎动物中发现的一种新型蛋白复合物。STN1和TEN1是酵母蛋白的同源物，参与端粒维持和末端保护。在人类中，CST被证明定位于端粒。人类CST亚基的损耗导致基因组不稳定的标志，即3H2AX灶和染色质桥增加，以及端粒异常，如g悬垂增加和端粒信号丢失。几个实验室的结果表明，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。本研究的初步结果表明，CST在端粒复制和复制叉重新启动中起作用。我们提出CST招募DNA pol - 1在难以复制的DNA位点重新启动DNA合成，这可能会阻碍DNA复制。在目标1中，CST在端粒复制中的作用将被研究。研究将进一步扩展，以确定是否需要CST来维持染色体脆弱位点。最后，将测量CST与RecQ解旋酶、WRN和BLM之间的相互作用在多大程度上需要维持端粒和脆弱位点的稳定性。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子在叉子失速后重新启动和单链DNA的水平。然后将探讨CST与复制叉组件和叉重新启动因子的相互作用。总之，这些研究将提供对CST如何通过难以复制的DNA帮助维持基因组稳定性的理解。