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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

基本信息

批准号：
9134830
负责人：
Jason Aaron Stewart
金额：
$ 24.9万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-06 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9134830
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 Fragile Sites Chromosome Segregation Chromosomes Complex DNA DNA Damage DNA Primase DNA Replication Factor DNA Sequence DNA Sequence Alteration DNA biosynthesis DNA lesion DNA polymerase alpha-primase DNA replication fork DNA-Binding Proteins Defect Disease Educational process of instructing Ensure Event Fiber Gene Duplication Genetic Transcription Genome Genomic DNA Genomic Instability Goals Head Hereditary Disease Human Human Genome K-Series Research Career Programs Knowledge Laboratories Lead Learning 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 Dynamics Proteins Protocols documentation Public Health RNA Research Research Proposals Role S Phase Sister Chromatid Site Specificity Techniques Telomere Maintenance Time Training Trinucleotide Repeats Universities Work base cancer genetics cancer initiation career development chromosome fusion cohesin cohesion daughter cell experience fusion gene human disease innovation insight live cell imaging member novel prevent protein function repaired replication factor A research study response telomere

项目摘要

Project Summary 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 α- primase (pol α) accessory factors, which stimulate pol α 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 α 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 chromatid 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.

项目概要我们目前对 DNA 复制如何进行的理解存在根本性差距自然发生的障碍以及 DNA 复制过程中姐妹染色单体内聚力 (SCC) 是如何建立的。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。基于由于人类基因组的大小，复制体复合物预计会在自然障碍下多次停滞整个S期过程。由于这些自然障碍不需要修复，因此细胞已经进化防止这些障碍引起 DNA 损伤反应和细胞周期停滞的机制。然而，这种现象是如何发生的仍然知之甚少。该项目的长期目标是阐明 DNA 复制是如何进行的穿过天然染色体屏障，例如重复 DNA 序列和 DNA 结合蛋白。这该职业发展奖的目标有两个：1）完成研究计划的具体目标，其中确定 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、获得职业发展在该奖项的 K99 阶段有教学经验并接受指导。 K99阶段将会发生在辛辛那提大学 Carolyn Price 博士的指导和额外支持下来自博士。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天然障碍包括难以复制的 DNA 区域，例如如端粒、脆弱位点、三核苷酸重复序列和着丝粒 DNA。在这些区域，复制体必须是停顿后重新启动。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，我的初步发现表明新发现的端粒相关 CST 复合物并不不仅在端粒上起作用，而且在 DNA 复制重启和 SCC 中也起作用。有趣的是，几个耗尽其他 DNA 复制蛋白会导致 SCC 缺陷和 DNA 复制重新启动，这表明两者之间存在联系这两个过程。 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 结构上与复制/修复因子复制蛋白 A (RPA) 相似。综合起来，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。这个假设的中心假设建议认为，CST 通过促进快速复制重启和 SCC 来防止基因组不稳定。难以复制的 DNA，例如端粒和脆弱位点。拟议的研究将解决这一假设通过三个具体目标： 1) 确定 CST 在 fork 停顿后促进复制重新启动的机制； 2) 阐明CST在姐妹染色单体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。在第一个目标中，CST 在复制重启中的作用将是通过分析 CST 耗尽细胞系中细胞和分子水平的重启进行研究，确定 CST 是否位于叉停顿位点，分析 CST 耗尽细胞系中的复制叉停顿难以复制的 DNA 位点并表征 CST ssDNA 结合活性。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，聘请一名在停滞的复制叉上分离 DNA 的新方案，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损失的时间和有丝分裂进展缺陷是否是由 CST 耗尽细胞中 SCC 丢失引起的。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 Prasad 博士合作 Jallepalli，纪念斯隆-凯特琳癌症中心的准会员和实验室负责人，也是该领域的专家染色体凝聚和有丝分裂。第三个目标将采用多管齐下的方法来确定 CST 的相互作用合作伙伴。这些研究将包括假设驱动的实验，以确定 CST 与蛋白质的相互作用参与 DNA 复制重启和 SCC。 CST 下拉后进行质谱分析将用作通过鉴定新型相互作用肽来深入了解 CST 功能的公正方法。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）它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。工作是意义重大，因为它将揭示染色体不稳定的一些潜在机制。每次一个细胞分裂其 DNA 必须正确复制并维持 SCC，以确保正确的染色体分离子细胞。 DNA 复制或染色体凝聚力的缺陷会导致与以下相关的表型癌症发生，例如易位、缺失、染色体融合、基因复制和非整倍体。一些遗传性疾病（称为粘连病）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病的基础，Coats 加。这些研究的完成将增进我们对这些细胞过程的理解，并提供新的预防和治疗这些疾病的目标。