Single-molecule dissection of a tumor- and virus-suppressing Smc complex involved in genome maintenance

参与基因组维护的肿瘤和病毒抑制 Smc 复合物的单分子解剖

• 批准号: 10536179
• 负责人: Jeremy Tzu-Huai Chang
• 金额: $ 5.18万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-11 至 2026-07-10
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10536179
• 关键词: Address; Affect; Architecture; Atomic Force Microscopy; Behavior; Binding; Biological Assay; Biology; Body of uterus; Chromosomes; Clinical; Color; Complex; Confocal Microscopy; DNA; DNA Binding; DNA Repair; DNA Replication Factor; DNA biosynthesis; DNA replication fork; Data; Defect; Detection; Dissection; Doctor of Philosophy; Endometrial Carcinoma; Eukaryota; Exhibits; Family; Fellowship; Fluorescence; Fluorescence Microscopy; Genome; Genomic Instability; Goals; Health; Hepatitis B Virus; Human Papillomavirus; In Vitro; Individual; Knowledge; Label; Literature; Maintenance; Malignant - descriptor; Malignant Neoplasms; Measures; Mediator of activation protein; Mentorship; Microfluidics; Molecular; Molecular Machines; Monitor; Movement; Mutation; Oncogenic Viruses; Outcome; Physiological; Play; Polymerase; Positioning Attribute; Property; Publishing; Reaction; Repression; Research; Research Personnel; Resolution; Role; Scientist; Single-Stranded DNA; Students; System; Technology; Testing; Time; Training; Tumor Suppression; Tumor Suppressor Proteins; Universities; Virus; Virus Diseases; Work; base; biophysical properties; career; cohesin; combinatorial; condensin; ds-DNA; experimental study; extrachromosomal DNA; genome integrity; helicase; human disease; in vivo; innovation; insight; laser tweezer; melting; member; novel strategies; novel therapeutic intervention; operation; optical traps; preference; programs; protein complex; reconstitution; replication stress; single molecule; therapeutic target; translocase; tumor

Project Summary/Abstract The structural maintenance of chromosomes (Smc) 5/6 complex plays a critical role in tumor suppression and the repression of tumor-causing viruses, such as the hepatitis B virus. Smc5/6 exerts these clinical functions by promoting faithful genome replication, coordinating DNA repair, and silencing extra-chromosomal DNA. However, there is little understanding of how Smc5/6 operates as a molecular machine, hindering our ability to intervene in Smc5/6’s health-related functions. Our central hypothesis is that Smc5/6 operates as a dynamic molecular machine that compacts DNA, intrinsically binds to DNA fork junctions, and co-localizes with replication factors. The long-term goal of this research is to understand how defects in Smc5/6 promote genome instability and malignant transformation. This project’s immediate objective is to elucidate the biophysical properties of the Smc5/6 complex by utilizing correlative single-molecule fluorescence and force microscopy, which combines optical tweezers, automated microfluidics, and multi-color confocal microscopy. In Specific Aim 1, Smc5/6’s DNA compaction abilities will be assayed on individual DNA tethers. The effect of the subunits of Smc5/6 and ATP will be systematically tested. The outcome of this work will define the role of each of these components on Smc5/6’s DNA compaction behavior. In Specific Aim 2, the binding behavior and dynamic movement of fluorescently-labeled Smc5/6 will be monitored on double-stranded DNA, single-stranded DNA, and fork junctions in real time. In Specific Aim 3, Smc5/6’s interactions with replication factors will be defined by a first-of-its-kind in vitro reconstitution of the eukaryotic replisome. Overall, this project will: (1) consolidate our understanding of Smc5/6 at the molecular level; (2) yield important insights into how eukaryotes maintain genome integrity and suppress tumors; and (3) potentiate new strategies to modulate Smc5/6’s physiological functions as a tumor suppressor and host restriction factor. Dr. Xiaolan Zhao, an expert on Smc5/6 biology who has a proven track-record for training successful scientists, and Dr. Shixin Liu, an expert on single-molecule technology who practices active mentorship, are co-sponsoring this proposal. The research efforts will take place at the Rockefeller University within the deeply supportive Tri-Institutional MD- PhD Program. This proposal and fellowship is an important career milestone for dual-degree students seeking to become independent investigators.

项目总结/摘要 染色体（Smc）5/6复合体的结构维持在肿瘤抑制和肿瘤生长中起关键作用。 抑制肿瘤病毒，如B型肝炎病毒。Smc 5/6通过以下方式发挥这些临床功能： 促进忠实的基因组复制、协调DNA修复和沉默染色体外DNA。 然而，人们对Smc 5/6如何作为分子机器运作的了解很少，这阻碍了我们研究Smc 5/6的能力。 干预Smc 5/6的健康相关功能。我们的中心假设是Smc 5/6作为一个动态的 压缩DNA的分子机器，本质上与DNA叉连接结合，并与复制共定位 因素这项研究的长期目标是了解Smc 5/6缺陷如何促进基因组不稳定性。 和恶性转化。 该项目的直接目标是通过利用Smc 5/6复合物来阐明Smc 5/6复合物的生物物理性质。 相关的单分子荧光和力显微镜，结合光镊，自动化 微流体和多色共聚焦显微镜。在特定目标1中，Smc 5/6的DNA压缩能力将是 对单个DNA链进行分析。将系统地测试Smc 5/6亚基和ATP的作用。 这项工作的结果将确定这些成分中的每一个对Smc 5/6的DNA压缩的作用 行为在特异性目标2中，荧光标记的Smc 5/6的结合行为和动态运动将 在双链DNA、单链DNA和分叉点上进行真实的实时监测。在具体目标3中， smc 5/6与复制因子的相互作用将通过首次在体外重建smc 5/6的表达来确定。 真核复制体 总体而言，本项目将：（1）巩固我们对Smc 5/6在分子水平上的理解;（2）产生重要的 深入了解真核生物如何保持基因组完整性和抑制肿瘤;（3）加强新的策略 调节Smc 5/6作为肿瘤抑制因子和宿主限制因子的生理功能。赵小兰博士， Smc 5/6生物学专家，在培养成功科学家方面有着良好的记录，刘世新博士， 一位积极指导的单分子技术专家，共同发起了这一提案。的 研究工作将在洛克菲勒大学进行，在三机构的大力支持下， 博士课程。这项建议和奖学金是一个重要的职业里程碑双学位学生寻求 成为独立调查员。