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

项目摘要/摘要 染色体的结构维持(SMC)5/6复合体在肿瘤抑制和转移中起着关键作用。 抑制引起肿瘤的病毒，如乙肝病毒。SMC5/6通过以下途径发挥这些临床功能 促进忠实的基因组复制，协调DNA修复，沉默染色体外DNA。 然而，人们对Smc5/6是如何作为分子机器运行的知之甚少，这阻碍了我们 干预SMC5/6‘S的健康相关功能。我们的中心假设是Smc5/6以动态的方式运行 一种分子机器，使DNA紧凑，固有地与DNA分叉连接结合，并与复制共同定位 各种因素。这项研究的长期目标是了解Smc5/6的缺陷如何促进基因组的不稳定性。 和恶变。 该项目的近期目标是通过利用Smc5/6复合体 相关的单分子荧光和力显微镜，结合了光镊子，自动化 微流体学和多色共聚焦显微镜。在具体目标1中，SMC5/6‘S的DNA紧凑能力将被 在单独的DNA系链上进行检测。Smc5/6和ATP亚单位的作用将被系统地测试。 这项工作的结果将确定这些成分中的每一个在SMC5/6‘S DNA压缩中的作用 行为。在特定目标2中，荧光标记的Smc5/6的结合行为和动态运动将 实时监测双链DNA、单链DNA和叉状连接。在具体目标3中， SMC5/6‘S与复制因子的相互作用将通过首例SMC5/6’SMC6与复制因子的体外重组来确定 真核复制体。 总体而言，这个项目将：(1)在分子水平上巩固我们对SMC5/6的理解；(2)产生重要的 对真核生物如何维持基因组完整性和抑制肿瘤的洞察；以及(3)加强新策略 调节SMC5/6‘S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， 一位Smc5/6生物学专家，在培养成功科学家方面有公认的记录，以及刘世新博士， 一位实行积极指导的单分子技术专家共同发起了这项提议。这个 洛克菲勒大学的研究工作将在深受支持的三机构MD- 博士学位课程。这一提议和奖学金是双学位学生寻求职业生涯的一个重要里程碑。 成为独立调查人员。

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.