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

基本信息

批准号：
10666417
负责人：
Jeremy Tzu-Huai Chang
金额：
$ 5.27万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
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/10666417
关键词：
Address Affect Architecture Atomic Force Microscopy Behavior Binding Biological Assay Biology Body of uterus Chromosomes Clinical Collaborations Color Complex Confocal Microscopy DNA DNA Repair DNA Replication Factor 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 Institution Knowledge Label Literature Maintenance Malignant - descriptor Malignant Neoplasms Measures Mediator Mentorship Microfluidics Molecular Molecular Machines Monitor Movement Mutation 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 barrier to testing 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 molecular dynamics novel strategies novel therapeutic intervention operation optic trap optic tweezer optical traps preference programs protein complex reconstitution replication stress restraint single molecule therapeutic target translocase tumor

项目摘要

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.

项目摘要/摘要染色体的结构维持（SMC）5/6复合物在肿瘤抑制和引起肿瘤病毒的表达，例如乙型肝炎病毒。 SMC5/6通过促进忠实的基因组复制，协调DNA修复和沉默的外染色体DNA。但是，对SMC5/6作为分子机的运行方式几乎没有理解，阻碍了我们的能力干预SMC5/6的健康相关功能。我们的中心假设是SMC5/6作为动态压缩DNA，本质上结合DNA叉连接的分子机，并与复制共定位因素。这项研究的长期目标是了解SMC5/6中缺陷如何促进基因组不稳定性和恶性转变。该项目的直接目标是通过使用SMC5/6复合物的生物物理特性相关的单分子荧光和力显微镜结合了光学镊子，自动化微流体和多色共聚焦显微镜。在特定的目标1中，SMC5/6的DNA压实能力将是在单个DNA系上进行测定。 SMC5/6和ATP亚基的影响将进行系统测试。这项工作的结果将定义这些组件在SMC5/6的DNA压实中的作用行为。在特定的目标2中，荧光标记的SMC5/6的结合行为和动态运动将会在双链DNA，单链DNA和叉子连接处进行监测。在特定的目标3中 SMC5/6与复制因素的相互作用将由首先的体外重构定义真核生物复制体。总体而言，该项目将：（1）巩固我们在分子水平上对SMC5/6的理解；（2）产生很重要深入了解真核生物如何保持基因组完整性并抑制肿瘤；（3）潜在的新策略将SMC5/6的物理功能调节为肿瘤抑制和宿主限制因子。小赵博士， SMC5/6生物学专家，拥有培训成功科学家的良好田径记录，以及Shixin Liu博士，实践积极精神训练的单分子技术专家正在共同发起该建议。研究工作将在洛克菲勒大学（Rockefeller University 博士计划。对于寻求双学位学生来说，这项建议和奖学金是一个重要的职业里程碑成为独立调查员。