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: 8386080
• 负责人: Jason Aaron Stewart
• 金额: $ 1.72万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386080
• 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.

描述（由申请人提供）：该项目的总体目标是通过难以复制的DNA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，例如端粒和脆弱的位点。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械的自然障碍。脆弱的位点表示为在复制应激条件下发生的染色体DNA中的间隙或断裂。已知端粒和脆弱的位点会引起基因组不稳定性，并在无法正确维持时会导致癌变。 CTC1-STN1-TEN1（CST）是一种新型的蛋白质复合物，最近在植物和脊椎动物中发现。 STN1和TEN1是酵母蛋白的同源物，与端粒维持和终端保护有关。在人类中，CST被证明与端粒本地化。人类CST亚基的耗竭导致基因组不稳定性的标志，即增加3H2AX灶和染色质桥，以及端粒异常，例如G-Overhangs和端粒信号损失增加。来自多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-蛋白酶（POL 1）相互作用，该酶启动DNA合成。该提案中提出的初步结果表明，CST在端粒复制和重复叉重新定叉中起作用。我们建议CST募集DNA POL 1来重新启用DNA到更复式的DNA位点的DNA合成，这可能会使DNA复制失速。在AIM 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维持染色体脆弱的位点。最后，将需要测量CST与RECQ解旋酶之间的相互作用，以维持端粒和脆弱位点稳定性。在AIM 2中，将研究CST促进复制叉进程的机制。叉车失速后的叉子和单链DNA的水平将在CST缺乏的细胞系中进行测量。将探索与复制叉组件和叉子重新激活因子的CST相互作用。这些研究将共同​​了解CST如何通过难以重复的DNA来帮助维持基因组稳定性。