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: 8896187
• 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
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-06 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8896187
• 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-Binding Proteins; DNA-Directed DNA Polymerase; Defect; Disease; Educational process of instructing; Ensure; Event; Fiber; Gene Duplication; Gene Mutation; Genetic Transcription; Genome; Genomic DNA; Genomic Instability; 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 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; repaired; replication factor A; research study; response; telomere

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.

项目摘要 我们目前对DNA复制如何通过 在DNA复制过程中，自然发生的障碍以及如何建立姐妹染色单体（SCC）。 在复制基因组期间，复制体综合体遇到了各种障碍。这些包括 不自然和自然的障碍。不自然的障碍包括DNA病变和双链断裂。 自然障碍包括重复的DNA，结合DNA的蛋白和RNA转录位点。基于 人类基因组的大小，复制体复合物预计会在自然障碍处多次停滞 通过S期。由于这些自然障碍不需要维修，因此细胞已经发展 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， 这种情况的发生方式仍然很糟糕。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（例如重复的DNA序列和DNA结合蛋白）进行进行。 该职业发展奖的目标是两个方面：1）完成研究建议的具体目的， 确定CTC1-STN1-TEN1（CST）复合物在DNA复制和SCC中的非细胞组作用， 2）通过学习新技术，开发一个独立的项目，获得成就，获得职业发展 在此奖项的K99阶段，教学经验并获得指导指导。 K99阶段将发生 在辛辛那提大学卡罗琳·普莱斯（Carolyn Price）博士的指导和指导下 来自博士。 Paul Chastain，David Kaufman，Prasad Jallepalli和Birgit Ehmer博士。 我们基因组中的自然障碍，即失速复制包括难以复制的DNA区域 作为端粒，脆弱的位点，三核苷酸重复序列和丝粒DNA。在这些地区，复制体必须是 失速后重新启动。但是，在这些位点如何重新启用DNA合成的表征仍然很差。 SCC是在DNA复制过程中建立的，并提议与复制体的通过同时发生。 令人惊讶的是，我的初步发现表明，新发现的，与端粒相关的CST综合体 仅在端粒功能，但在DNA复制中也重新启动和SCC。有趣的是，几个 其他DNA复制蛋白会导致SCC和DNA复制的缺陷重新启动，这表明 这两个过程。 CST，CTC1和STN1的两个成分最初被鉴定为DNA聚合酶α- 刺激polα结合和原始酶活性的原始酶（polα）辅助因子。 CST还结合了ssDNA 在结构上与复制/修复因子复制蛋白A（RPA）相似。在一起，这些发现 表明CST与Polα的相互作用对于其非telomere功能很重要。中心假设 提案是CST通过促进在 难以重复的DNA，例如端粒和脆弱的位点。拟议的研究将解决这一假设 通过三个特定的目的：1）确定叉子失速后CST收藏夹复制重新启动的机制； 2）阐明CST在姐妹染色单体内聚和有丝分裂进程中的作用； 3）识别与CST的相互作用 复制重新启动和姐妹染色单体内聚因子。在第一个目标中，CST在复制重新启动中的作用将是 通过分析在CST缺乏的细胞系中分析在细胞和分子水平上重新启动的研究，从而确定 CST是否本地化与叉子停滞的位点，分析在CST缺乏的细胞系中的复制叉处。 难以重复DNA并表征CST ssDNA结合活性的位点。要执行这些实验， 我将接受北卡罗来纳大学Paul Chastain博士的DNA纤维分析培训，员工A 在停滞的复制叉上隔离DNA的新方案，并利用我的生化和细胞生物学培训。在 第二个目的，CST在SCC中的作用将通过首先确定凝聚力损失的时机和 是否由CST缺乏的细胞中的SCC丢失引起有丝分裂进展的缺陷。这些研究将要求我 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是纪念斯隆凯特癌中心的副成员兼实验室负责人，也是专家 染色体内聚和有丝分裂。第三个目标将使用多管齐下的方法来确定CST相互作用 合作伙伴。这些研究将包括假设驱动的实验，以鉴定CST与蛋白质的相互作用 参与DNA复制重新启动和SCC。 CST下拉，然后将质谱法用作 通过鉴定新型相互作用的肽来洞悉CST功能的无偏方法。 这项提出的工作具有创新性，因为：1）它解决了CST意外的非居组功能； 2） 它研究了在叉子停滞在自然障碍处后重新定义DNA合成的新机制。 3）它结合了各种新的良好的技术来研究中心假设。工作是 意义重大，因为它会揭示染色体不稳定性的某些潜在机制。每次一个单元 必须正确复制其DNA并保持SCC，以确保正确的染色体隔离到 子细胞。 DNA复制或染色体内聚力的缺陷导致与表型相关的表型 癌症开始，例如易位，缺失，染色体融合，基因重复和非整倍性。 遗传疾病，称为内介型，也与SCC丧失和染色体破裂有关。 此外，最近显示CTC1中的突变是罕见的常染色体隐性疾病的基础 加。这些研究的完成将提高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靶标。