How bacterial SMC complexes organize chromosomes

项目摘要 复制染色体的组织和分离是生命系统的基础。结构性 染色体的维持(SMC)复合体在生命的所有领域中都在这些过程中发挥着核心作用。 这些环状ATPase共享共同的结构和亚基间的接触，与共同的 作用机制。最近对枯草芽孢杆菌和真核生物的研究提供了令人信服的体内和 SMC复合体利用ATP水解物挤出DNA环的体外证据。然而，目前还不清楚是如何 当染色体被大量蛋白质包裹并与之作用时，SMC复合体会挤出DNA环 复制和转录机器。我的研究小组的目标是确定SMC复合体在 它们会遇到复制体或其他凝集素分子。我们还将描述SMC复合体是如何 当它们到达终点区时从染色体上释放。最后，我们将探讨SMC在 同时包含环状和线状结构的多部分细菌基因组的组织和动力学 染色体。总而言之，我们的工作有可能提供染色体折叠的一般原则 在所有的有机体中都是紧凑的。 在这份申请中，我申请了一项行政补助，以支持一名本科生 在我的实验室，林赛女士，2023年夏天，在我的实验室做一个研究项目。这样做的目的是 该项目旨在支持MS Way在暑期全职研究细菌细胞生物学和分子方面的经验 生物学。韦女士目前是路易斯安那州立大学的大三学生。她决心攻读生物医学博士学位。一个完整的- 今年夏天的时间研究将更好地为她的研究生院做准备。韦女士的项目将调查 SMC复合体与细菌和古生物组蛋白样蛋白之间的相互作用， 包括来自大肠杆菌的HupA和H-NS，以及来自费氏甲烷杆菌的HMfA和HMFB。威尔女士：1) 构建表达融合到mCherry上的这些蛋白的表达载体和菌株；2)用 荧光显微镜；3)用Western blotting定量它们的表达；4)监测 通过全基因组染色体构象捕捉Hi-C分析SMC与组蛋白的相互作用。韦女士将 出席我们每周的实验室会议。她还将在印第安纳大学微生物学杂志上展示一张海报 2023年10月的年度务虚会。

Project Summary The organization and segregation of replicated chromosomes are fundamental to living systems. Structural maintenance of chromosomes (SMC) complexes play central roles in these processes in all domains of life. These ring-shaped ATPases share common structures and inter-subunit contacts, consistent with a common mechanism of action. Recent studies in Bacillus subtilis and eukaryotes have provided compelling in vivo and in vitro evidence that SMC complexes utilize ATP hydrolysis to extrude DNA loops. However, it is unclear how SMC complexes extrude DNA loops when the chromosome is coated by numerous proteins and acted upon by replication and transcription machineries. My research group aims to determine how SMC complexes act when they encounter the replisome or other condensin molecules. We will also characterize how SMC complexes are released from the chromosome when they reach the terminus region. Finally, we will explore SMC’s role in the organization and dynamics of a multipartite bacterial genome that contains both a circular and a linear chromosome. Taken together, our work has the potential to provide the general principles of chromosome folding and compaction in all organisms. In this proposed application, I am requesting an administrative supplement to support an undergraduate in my lab, Ms. Lindsey Way, to do a research project in my lab during the summer of 2023. The goal of this project is to support Ms. Way for full-time summer research experience in bacterial cell biology and molecular biology. Ms. Way is currently a Junior at IU. She is determined to pursue a Ph.D. in biomedical sciences. A full- time research in this summer will better prepare her for graduate school. Ms. Way’s project will investigate the interactions between SMC complexes and a collection of histone-like proteins from bacteria and archaea, including HupA and H-NS from E. coli, and HMfA and HMfB from Methanothermus fervidus. Ms. Way will: 1) build plasmids and strains to express these proteins fused to mCherry; 2) visualize these fusion proteins using fluorescence microscopy; 3) quantify their expression using Western blotting; 4) monitor the consequences of the SMC-histone interactions by genome-wide chromosome conformation capture Hi-C assays. Ms. Way will present in our weekly lab meetings. She will also present a poster in the Indiana University Microbiology Annual Retreat in October 2023.