CAREER: Conformational dynamics of DNA and nucleosomes: A quantitative single-molecule study

职业：DNA 和核小体的构象动力学：定量单分子研究

• 批准号: 0644414
• 负责人: Marcia Levitus
• 金额: $ 56.9万
• 依托单位: Arizona State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0644414&HistoricalAwards=false
• 关键词: CAREER; Conformational; dynamics; DNA; nucleosomes

This project is directed toward achieving a quantitative understanding of the dynamical processes that occur in DNA and nucleosomes. Nucleosomes are the first and most fundamental packing level of genomic DNA in eukaryotes, which consist of a complex of eight histone proteins around which 147 base pairs of DNA are wrapped in approximately two superhelical turns. Nucleosomal organization protects genomic DNA from enzymatic degradation, but it also makes most of the genome inaccessible to the many proteins that must bind to it for gene regulation, transcription, replication, recombination, and repair. The elucidation of the mechanisms and rates of the spontaneous conformational changes in nucleosomal DNA is critical to understand how such proteins can gain access to their target sites without requiring the complete dissociation of the histone octamer. The specific aims of the project seek to gain a deeper quantitative understanding of the simplest biological components of chromatin: double stranded DNA and nucleosomes. A new experimental approach based on fluorescence fluctuation spectroscopy will allow the determination of the rates of conformational changes in biopolymers in a broad range of timescales. These, and other methods of analysis, will be applied to the study of DNA flexibility and dynamics in naked DNA and nucleosomes. The proposed approach is novel, in that it has a strong emphasis in the application and development of new methods of data acquisition and analysis that will advance the single-molecule field towards a more quantitative era. The successful completion of these studies is expected to establish the foundation necessary to undertake the study of the more complex biological systems such as native chromatin.This proposal involves research and teaching activities that aim to stress the importance of quantitative research. The lack of basic numerical skills, and the negative attitude that many students hold about mathematics, conflicts with the demands of today's interdisciplinary research and seriously limits students in their long-term opportunities. The PI proposes to develop a course intended to act as a bridge between the traditional math classes and the more quantitative science courses. The PI proposes to create a website that will gather testimonials and information of faculty members around the nation illustrating 1) the need of learning the quantitative sciences in today's biologically-oriented disciplines, and 2) the challenges of being a minority in science. The latter will be bilingual English/Spanish, and will be written in lay terms with the goal of reaching not only students, but also their families and the general public.

该项目旨在实现对DNA和核小体中发生的动力学过程的定量理解。 核小体是真核生物中基因组DNA的第一个也是最基本的包装水平，它由8个组蛋白组成的复合物组成，147个碱基对的DNA被包裹在大约两个超螺旋圈中。 核小体组织保护基因组DNA免受酶促降解，但它也使大多数基因组无法接近许多蛋白质，这些蛋白质必须与其结合以进行基因调控、转录、复制、重组和修复。阐明核小体DNA中自发构象变化的机制和速率对于理解这些蛋白质如何能够在不需要组蛋白八聚体完全解离的情况下进入其靶位点是至关重要的。 该项目的具体目标是寻求对染色质最简单的生物成分：双链DNA和核小体有更深入的定量了解。 一种新的实验方法的基础上荧光波动光谱将允许在很宽的时间尺度范围内的生物聚合物的构象变化率的测定。 这些和其他分析方法将被应用于裸DNA和核小体中DNA柔性和动力学的研究。所提出的方法是新颖的，因为它非常强调数据采集和分析的新方法的应用和开发，这将推动单分子领域走向更加定量的时代。这些研究的成功完成将为研究更复杂的生物系统（如天然染色质）奠定必要的基础。本提案涉及旨在强调定量研究重要性的研究和教学活动。缺乏基本的数学技能，以及许多学生对数学的消极态度，与当今跨学科研究的要求相冲突，严重限制了学生的长期机会。PI建议开发一门课程，旨在作为传统数学课程和更量化的科学课程之间的桥梁。 PI建议创建一个网站，收集全国各地教师的推荐和信息，说明1）在当今以生物学为导向的学科中学习定量科学的必要性，以及2）在科学中成为少数民族的挑战。后者将是双语英语/西班牙语，并将在非专业术语写，不仅达到学生，而且他们的家庭和公众的目标。