Visualizing the Dynamics of Chromatin and Chromatin Remodeling Proteins

描述（由申请人提供）：DNA复制、转录、修复、表观遗传和染色体分离都是维持细胞活力的关键过程。在真核生物中，这些功能必须在组织成高度浓缩的染色质的DNA上执行。随着越来越多的疾病相关基因的发现，染色质在人类疾病中的重要性已经变得非常清楚，一些疾病已经与染色质生物学缺陷有关。为了帮助理解DNA代谢的不同方面如何受到染色质的影响，我们开发了独特的技术，使我们能够使用光学显微镜在单分子水平上直接可视化数百个单个DNA分子。在这里，我们将通过使用单分子光学显微镜实时可视化这些过程，评估核小体如何影响与DNA代谢相关的反应的空间和时间进展。我们将分析影响核小体定位的因素，我们将确定核小体如何影响DNA修复蛋白，我们将询问核小体如何受到与DNA运动蛋白相互作用的影响，并且我们将开始努力对表观遗传现象进行机制理解。我们将寻求确定与这些问题相关的详细机制信息，这个项目的部分意义在于我们努力获得的答案的深度。

DESCRIPTION (provided by applicant): DNA replication, transcription, repair, epigenetic inheritance, and chromosome segregation are all processes critical for maintaining cellular viability. In eukaryotes, these functions must be carried out on DNA that is organized into highly condensed chromatin. With the identification of an increasing number of disease-associated genes, the importance of chromatin in human disorders has become abundantly clear, and several diseases have been linked to defects in chromatin biology. To help understand how different aspects of DNA metabolism are influenced by chromatin we have developed unique technologies that allow us to directly visualize hundreds of individual DNA molecules at the single molecule level using optical microscopy. Here we will assess how nucleosomes influence the spatial and temporal progression of reactions related to DNA metabolism by visualizing these processes in real time using single molecule optical microscopy. We will analyze factors that influence nucleosome positioning, we will determine how nucleosomes affect DNA repair proteins, we will ask how nucleosomes are affected by interactions with DNA motor proteins, and we will begin working towards a mechanistic understanding of epigenetic phenomena. We will seek to determine detailed mechanistic information related to these questions, and part of the significance of this project lies in the depth of the answers we strive to obtain.