Division of Molecular and Cellular Biosciences: Investigator-initiated research projects (MCB)

分子和细胞生物科学部：研究者发起的研究项目（MCB）

• 批准号: 1148818
• 负责人: Joseph Loparo
• 金额: $ 62.43万
• 依托单位: Harvard University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1148818&HistoricalAwards=false
• 关键词: Division; Molecular; Cellular; Biosciences; Investigator

Intellectual meritBacteria typically store their genetic information in a single circular chromosome that is several million DNA bases long. In order to maintain and duplicate this chromosome, called the nucleoid, bacteria must accomplish two major feats of structural engineering: First, a giant 1.5 millimeter-long DNA molecule must be packaged into a bacterial cell that is over a thousand times shorter. Second, newly replicated sister chromosomes must be disentangled and separated in preparation for cell division. Work over the last several decades has identified a number of nucleoid-associated proteins (NAPs) that play essential roles in these processes, yet it remains unclear how various NAP-DNA interactions collectively regulate nucleoid architecture. Using single-molecule biochemical assays, this project aims to investigate the molecular mechanisms by which nucleoid-associated proteins from the model bacterium Bacillus subtilis organize and segregate the bacterial chromosome. This study will focus on five key NAPs (Spo0J, HBsu, SASP, Rok and SMC) selected due to their essential functions throughout different parts of the B. subtilis life cycle. To develop a mechanistic understanding of how these NAPs function, a novel single-molecule assay will be employed that correlates NAP-DNA binding with changes in the elastic properties of individual DNA molecules. These techniques will be applied to address the following aims: (1) To determine how each of the individual NAPs associates with DNA and modulates its mechanical properties. (2) To establish how HBsu, the major non-specific NAP in B. subtilis, facilitates or antagonizes the interactions of Spo0J, SASP and Rok with DNA. (3) To characterize the SMC complex and establish if DNA compaction is driven by intra- or intermolecular SMC interactions. These studies will take the first steps toward reconstituting and ultimately dissecting the complexity of the chromosome by investigating cooperativity between NAPs, while the techniques developed will be broadly applicable to other studies of protein-DNA interactions.Broader ImpactsThe single-molecule methods employed in this project provide a quantitative description of the dynamics of proteins and biological macromolecules, such as DNA and RNA, yet they have not gained popularity in the classroom because they are regarded as too difficult and expensive. In order to increase accessibility, an inexpensive single-molecule microscope will be designed that can be used in the undergraduate and high school teaching laboratory. A suite of experiments will be developed for this microscope to introduce biophysical concepts such as Brownian motion, DNA polymer dynamics and the principles of molecular motors. These experiments will be piloted in an undergraduate laboratory course and introduced to high school teachers and students through summer workshops in which the PI will participate.

细菌通常把它们的遗传信息储存在一条有几百万个DNA碱基长的圆形染色体上。为了维持和复制这种被称为类核的染色体，细菌必须完成两项主要的结构工程壮举：首先，必须将一个巨大的1.5毫米长的DNA分子包装到一个比它短一千多倍的细菌细胞中。其次，新复制的姐妹染色体必须解开并分离，为细胞分裂做准备。过去几十年的工作已经确定了许多在这些过程中起重要作用的核相关蛋白（nap），但仍不清楚各种NAP-DNA相互作用如何共同调节类核结构。利用单分子生化分析，本项目旨在研究模式细菌枯草芽孢杆菌核相关蛋白组织和分离细菌染色体的分子机制。本研究将重点研究枯草芽孢杆菌在不同生命周期中发挥重要作用的5个关键nap （Spo0J、HBsu、SASP、Rok和SMC）。为了进一步了解这些nap的功能机制，将采用一种新的单分子分析方法，将NAP-DNA结合与单个DNA分子弹性特性的变化联系起来。这些技术将用于解决以下目标：(1)确定每个单独的nap如何与DNA结合并调节其机械性能。(2)了解枯草芽孢杆菌中主要的非特异性NAP HBsu如何促进或拮抗Spo0J、SASP和Rok与DNA的相互作用。(3)表征SMC复合物，并确定DNA压实是否由分子内或分子间SMC相互作用驱动。这些研究将通过研究nap之间的协同作用，为重建和最终剖析染色体的复杂性迈出第一步，而所开发的技术将广泛适用于其他蛋白质- dna相互作用的研究。更广泛的影响本项目中采用的单分子方法提供了蛋白质和生物大分子（如DNA和RNA）动力学的定量描述，但由于它们被认为过于困难和昂贵，因此在课堂上并没有得到普及。为了增加可及性，将设计一种廉价的单分子显微镜，可用于本科和高中教学实验室。将为该显微镜开发一套实验，以介绍生物物理概念，如布朗运动，DNA聚合物动力学和分子马达原理。这些实验将在本科实验课程中进行试点，并通过PI将参加的暑期研讨会向高中教师和学生介绍。