Roles of the mammalian CST complex in DNA replication and chromosome cohesion

哺乳动物 CST 复合体在 DNA 复制和染色体凝聚中的作用

• 批准号: 8425980
• 负责人: Jason Aaron Stewart
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-06 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8425980
• 关键词: Address; Affect; Agreement; Anaphase; Aneuploidy; Award; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Cell Cycle Arrest; Cell Line; Cell physiology; Cells; Cellular biology; Chromosomal Instability; Chromosome Breakage; Chromosome Cohesion; Chromosome Deletion; Chromosome Fragile Sites; Chromosome Segregation; Chromosomes; Complex; DNA; DNA Damage; DNA Primase; DNA Replication Factor; DNA Sequence; DNA biosynthesis; DNA lesion; DNA polymerase alpha-primase; DNA-Binding Proteins; Defect; Disease; Educational process of instructing; Ensure; Event; Fiber; Gene Duplication; Gene Mutation; Genetic Transcription; Genome; Genomic Instability; Genomics; Goals; Head; Hereditary Disease; Human; Human Genome; K-Series Research Career Programs; Knowledge; Laboratories; Lead; Learning; Life; Link; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Memorial Sloan-Kettering Cancer Center; Mentors; Mentorship; Metaphase; Methods; Mitosis; Mitotic; Molecular; Mutation; North Carolina; Peptides; Phase; Phenotype; Play; Polymerase; Prevention; Price; Process; Protein Analysis; Protein Binding; Protein Dynamics; Proteins; Protocols documentation; Public Health; RNA; Research; Research Proposals; Role; S Phase; Sister; Sister Chromatid; Site; Specificity; Techniques; Telomere Maintenance; Time; Training; Trinucleotide Repeats; Universities; Work; base; cancer genetics; cancer initiation; career development; cellular imaging; cohesin; cohesion; daughter cell; experience; fusion gene; human disease; innovation; insight; member; novel; prevent; protein function; public health relevance; repaired; replication factor A; research study; response; telomere

DESCRIPTION (provided by applicant): There is a fundamental gap in our current understanding of how DNA replication proceeds through naturally occurring barriers and how sister chromatid cohesion (SCC) is established during DNA replication. During replication of the genome, the replisome complexes encounter a variety of barriers. These include unnatural and natural impediments. Unnatural impediments include DNA lesions and double-strand breaks. Natural impediments include repetitive DNA, DNA-bound proteins and sites of RNA transcription. Based on the size of the human genome, replisome complexes are predicted to stall many times at natural impediments throughout the course of S-phase. Since these natural impediments do not require repair, the cell has evolved mechanisms to prevent these impediments from causing a DNA damage response and cell cycle arrest. However, how this occurs remains poorly understood. The long-term goal of this project is to elucidate how DNA replication proceeds through natural chromosome barriers, such as repetitive DNA sequences and DNA-bound proteins. The objective of this career development award is two-fold: 1) complete the specific aims of the research proposal, which are to determine the non-telomere roles of the CTC1-STN1-TEN1 (CST) complex in DNA replication and SCC, and 2) receive career development through learning new techniques, developing an independent project, gaining teaching experience and receiving guided mentoring in the K99 phase of this award. The K99 phase will occur under the mentorship and guidance of Dr. Carolyn Price at the University of Cincinnati with additional support from Drs. Paul Chastain, David Kaufman, Prasad Jallepalli and Dr. Birgit Ehmer. Natural impediments in our genome that stall replication include difficult-to-replicate DNA regions such as telomeres, fragile sites, trinucleotide repeats and centromeric DNA. At these regions, the replisome must be restarted after stalling. However, how DNA synthesis is reinitiated at these sites remains poorly characterized. SCC is established during DNA replication and proposed to occur concurrent with passage of the replisome. Surprisingly, my preliminary findings suggest that the newly discovered, telomere-associated CST complex not only functions at the telomere but also in both DNA replication restart and SCC. Interestingly, depletion of several other DNA replication proteins leads to defects in SCC and DNA replication restart, suggesting a link between these two processes. Two components of CST, CTC1 and STN1, were originally identified as DNA polymerase alpha- primase (pol alpha) accessory factors, which stimulate pol alpha binding and primase activities. CST also binds ssDNA and is structurally similar to the replication/repair factor replication protein A (RPA). Together, these findings suggest that CST interactions with pol alpha are important for its non-telomere functions. The central hypothesis of this proposal is that CST prevents genome instability by promoting rapid replication restart and SCC at sites of difficult-to replicate DNA, such as telomeres and fragile sites. The proposed research will address this hypothesis through three specific aims: 1) To determine the mechanism by which CST facilitates replication restart after fork stalling; 2) To elucidate the role of CST in sister chroatid cohesion and mitotic progression; 3) To identify CST interactions with replication restart and sister chromatid cohesion factors. In the first aim, the role of CST in replication restart will be investigated by analyzing restart at both the cellular and molecular level in CST-depleted cell lines, determining whether CST is localized to sites of fork stalling, analyzing replication fork stalling in CST-depleted cell lines at sites of difficult-to-replicate DNA and characterizing CST ssDNA binding activity. To perform these experiments, I will receive training in DNA fiber analysis from Dr. Paul Chastain at the University of North Carolina, employ a new protocol for isolating DNA at stalled replication forks and utilize my biochemical and cell biology training. In the second aim, the role of CST in SCC will be assessed by first determining the timing of cohesion loss and whether defects in mitotic progression arise from SCC loss in CST-depleted cells. These studies will require me to learn live-cell imaging and new cell biology techniques. For these studies, I will be collaborating with Dr. Prasad Jallepalli, associate member and laboratory head at the Memorial Sloan-Kettering Cancer Center and an expert in chromosome cohesion and mitosis. The third aim will use a multi-pronged approach to determine CST interacting partners. These studies will include hypothesis-driven experiments to identify CST interactions with proteins involved in DNA replication restart and SCC. CST pull-down followed by mass spectrometry will be used as an unbiased approach to gain insight into CST function through the identification of novel interacting peptides. This proposed work is innovative because: 1) it addresses the unexpected non-telomere functions of CST; 2) it investigates novel mechanisms for the reinitiation of DNA synthesis after fork stalling at natural impediments; 3) it combines a variety of new and well-established techniques to investigate the central hypothesis. The work is significant because it will reveal some of the underlying mechanisms of chromosome instability. Each time a cell divides its DNA must be properly replicated and SCC maintained to ensure proper chromosome segregation to the daughter cells. Defects in either DNA replication or chromosome cohesion lead to phenotypes associated with cancer initiation, such as translocations, deletions, chromosome fusions, gene duplication and aneuploidy. Several genetic disorders, termed cohesionopathies, are also associated with SCC loss and chromosome breakage. Furthermore, mutations in CTC1 were recently shown to underlie a rare autosomal recessive disorder, Coats plus. The completion of these studies will advance our understanding of these cellular processes and provide new targets for prevention and treatment of these diseases.

描述（由申请人提供）：我们目前对 DNA 复制如何通过自然存在的障碍以及姐妹染色单体凝聚力 (SCC) 在 DNA 复制过程中如何建立的理解存在根本性差距。在基因组复制过程中，复制体复合物遇到各种障碍。这些包括非自然和自然障碍。非自然障碍包括 DNA 损伤和双链断裂。自然障碍包括重复 DNA、DNA 结合蛋白和 RNA 转录位点。根据人类基因组的大小，预计复制体复合物在整个 S 期过程中会多次因自然障碍而停滞。由于这些自然障碍不需要修复，细胞已经进化出机制来防止这些障碍引起 DNA 损伤反应和细胞周期停滞。然而，人们对这种现象如何发生仍知之甚少。该项目的长期目标是阐明 DNA 复制如何通过天然染色体屏障（例如重复 DNA 序列和 DNA 结合蛋白）进行。该职业发展奖的目标有两个：1) 完成研究计划的具体目标，即确定 CTC1-STN1-TEN1 (CST) 复合物在 DNA 复制和 SCC 中的非端粒作用，2) 通过学习新技术、开发独立项目、获得教学经验以及在该奖项的 K99 阶段接受指导来获得职业发展。 K99 阶段将在辛辛那提大学 Carolyn Price 博士的指导和指导下进行，并得到 Drs. Carolyn Price 的额外支持。保罗·查斯坦、大卫·考夫曼、普拉萨德·贾勒帕利和比尔吉特·埃默博士。 我们基因组中阻碍复制的自然障碍包括难以复制的 DNA 区域，例如端粒、脆弱位点、三核苷酸重复和着丝粒 DNA。在这些区域，复制体必须在停止后重新启动。然而，DNA 合成是如何在这些位点重新启动的仍然知之甚少。 SCC 是在 DNA 复制过程中形成的，并且与复制体的传递同时发生。令人惊讶的是，我的初步研究结果表明，新发现的端粒相关 CST 复合物不仅在端粒上发挥作用，而且还在 DNA 复制重启和 SCC 中发挥作用。有趣的是，其他几种 DNA 复制蛋白的消耗会导致 SCC 缺陷和 DNA 复制重新启动，这表明这两个过程之间存在联系。 CST 的两个成分 CTC1 和 STN1 最初被鉴定为 DNA 聚合酶 α-引物酶 (pol α) 辅助因子，可刺激 pol α 结合和引物酶活性。 CST 还结合 ssDNA，在结构上与复制/修复因子复制蛋白 A (RPA) 相似。总之，这些发现表明 CST 与 pol α 的相互作用对其非端粒功能很重要。该提案的中心假设是，CST 通过促进快速复制重启和难以复制的位点的 SCC 来防止基因组不稳定。 复制 DNA，例如端粒和脆弱位点。拟议的研究将通过三个具体目标来解决这一假设：1）确定 CST 在 fork 停顿后促进复制重新启动的机制； 2) 阐明CST在姐妹脉络凝聚和有丝分裂进展中的作用； 3) 确定 CST 与复制重启和姐妹染色单体凝聚因子的相互作用。在第一个目标中，CST 在复制重启中的作用将是 通过分析 CST 耗尽的细胞系中细胞和分子水平的重启、确定 CST 是否定位于叉停顿位点、分析 CST 耗尽的细胞系中难以复制 DNA 的复制叉停顿以及表征 CST ssDNA 结合活性来进行研究。为了进行这些实验，我将接受北卡罗来纳大学 Paul Chastain 博士的 DNA 纤维分析培训，采用新方案在停滞的复制叉上分离 DNA，并利用我的生化和细胞生物学培训。在 第二个目标是，通过首先确定内聚力丧失的时间以及有丝分裂进展的缺陷是否由 CST 耗尽的细胞中的 SCC 丧失引起，来评估 CST 在 SCC 中的作用。这些研究需要我学习活细胞成像和新的细胞生物学技术。对于这些研究，我将与纪念斯隆-凯特琳癌症中心的准成员兼实验室负责人、染色体凝聚和有丝分裂方面的专家 Prasad Jallepalli 博士合作。第三个目标将采用多管齐下的方式来确定 CST 的互动伙伴。这些研究将包括假设驱动的实验，以确定 CST 与参与 DNA 复制重启和 SCC 的蛋白质的相互作用。 CST 下拉后进行质谱分析将作为一种公正的方法，通过鉴定新型相互作用肽来深入了解 CST 功能。 这项拟议的工作具有创新性，因为：1）它解决了 CST 意想不到的非端粒功能； 2) 研究了在自然障碍处停顿后重新启动 DNA 合成的新机制； 3）它结合了各种新的和成熟的技术来研究中心假设。这项工作意义重大，因为它将揭示染色体不稳定的一些潜在机制。每次细胞分裂时，其 DNA 都必须正确复制并维持 SCC，以确保子细胞的染色体正确分离。 DNA复制或染色体凝聚力的缺陷会导致与癌症发生相关的表型，例如易位、缺失、染色体融合、基因复制和非整倍体。几种称为粘连病的遗传性疾病也与 SCC 丢失和染色体断裂有关。此外，CTC1 突变最近被证明是一种罕见的常染色体隐性遗传疾病 Coats plus 的基础。这些研究的完成将增进我们对这些细胞过程的理解，并为预防和治疗这些疾病提供新的目标。