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

这个项目旨在实现对DNA和核小体中发生的动态过程的定量了解。核小体是真核生物基因组DNA的第一个也是最基本的堆积水平，由8个组蛋白组成的复合体，其周围的147个碱基对的DNA被包裹在大约两个超螺旋旋转中。核小体组织保护基因组DNA免受酶降解，但它也使大多数基因组无法被许多蛋白质访问，这些蛋白质必须与核小体结合以进行基因调节、转录、复制、重组和修复。核小体DNA自发构象变化的机制和速率的阐明对于理解这些蛋白质如何在不需要完全解离组蛋白八聚体的情况下获得其靶点是至关重要的。该项目的具体目标是寻求更深入地定量了解染色质最简单的生物成分：双链DNA和核小体。基于荧光涨落光谱的一种新的实验方法将允许在广泛的时间范围内确定生物聚合物的构象变化速率。这些方法和其他分析方法将被应用于研究裸露DNA和核小体中的DNA灵活性和动力学。提出的方法是新颖的，因为它强调了新的数据采集和分析方法的应用和发展，这将把单分子领域推向更定量的时代。这些研究的成功完成可望为研究更复杂的生物系统，如天然色素奠定必要的基础。这项建议涉及旨在强调定量研究重要性的研究和教学活动。缺乏基本的数字技能，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，旨在充当传统数学课程和更多定量科学课程之间的桥梁。PI建议创建一个网站，收集全国各地教职员工的推荐信和信息，说明1)在当今以生物为导向的学科中学习定量科学的必要性，以及2)作为科学界的少数群体所面临的挑战。后者将是英语/西班牙语双语，并将以非专业术语编写，目的不仅是接触到学生，而且还包括他们的家人和普通公众。

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.