Single molecule and biophysical studies of nucleic acid remodeling

项目摘要 DNA和RNA的单分子测量及其相互作用和处理揭示了 这些分子的功能以及它们的活动是如何被调节的。我们之前对DNA的机制研究 解旋酶和CRISPR酶促使我们开发新的生物技术工具， 在体外和活细胞中。我们还采用了下一代测序技术， 用于大规模平行测量DNA力学和染色质重塑活性的生物物理测定 规模我们建议研究CRISPR-Cas9、染色质重塑和DNA错配的三个广泛领域。 及其修复。一个贯穿始终的主题是探索丰富的序列空间，而这些空间通常在 机械研究。 为了使用CRISPR-Cas9进行有效的基因组编辑，需要将Cas9核糖核蛋白复合物（RNP） 在高浓度下组装，需要指导RNA的精确共转录折叠及其 通过Cas9结合的稳定化。此外，在靶DNA切割后，Cas9-RNP需要快速解离，以用于切割。 细胞来检测损伤并产生DNA损伤反应。了解这两个关键步骤 由靶序列调控的基因将帮助研究人员优化靶位点选择和指导RNA设计。 染色质重塑剂是ATP酶，它通过以下方式改变核小体的位置或组成 核小体滑动或组蛋白交换。许多开创性的研究，包括那些使用单- 分子方法，揭示了滑动反应的细节，但我们对组蛋白的了解， 交换反应受到更大的限制。我们建议开发单分子测定法来完全确定 SWR 1催化H2 A-H2 B二聚体交换为H2 A. Z-H2 B二聚体的动力学和途径。我们还将 发现和表征酵母天然基因组序列，使核小体形成在一个明确的定义， 位置，我们希望它将取代人工核小体定位序列，'601'，已被用于 几乎所有涉及核小体的机制研究。 DNA错配修复是由保守的蛋白质机制进行的，但尽管经过了几十年的研究， 对不同序列背景下的不同错配如何修复的理解非常有限。 例如，大多数机制研究都是针对单一类型的错配（GT错配）进行的。我们 将开发一种高通量的方法来测量体内错配修复效率的数千 不同的错配如果未修复，直到组蛋白沉积在新生的DNA上，错配将成为一个基因组的一部分。 核小体我们将研究含有错配的核小体的生物物理特性及其与 染色质重塑，使我们能够研究如何未修复的错配可以影响DNA的可及性。

Project Summary Single-molecule measurements of DNA and RNA, and their interactions and processing have revealed how these molecules function and how their activities are regulated. Our previous mechanistic studies of DNA unwinding enzymes and CRISPR enzymes have motivated us to develop novel biotechnological tools applicable in vitro and in living cells. We have also adopted next generation sequencing to develop high throughput biophysical assays for measuring DNA mechanics and chromatin remodeling activities in a massively parallel scale. We propose to pursue three broad areas of CRISPR-Cas9, chromatin remodeling, and DNA mismatch and its repair. A permeating theme is to explore the rich sequence space that is often poorly sampled in mechanistic studies. For effective genome editing using CRISPR-Cas9, Cas9 ribonucleoprotein complexes (RNPs) need to be assembled in high concentrations, requiring accurate co-transcriptional folding of the guide RNA and its stabilization by Cas9 binding. In addition, after target DNA cleavage, a Cas9-RNP needs to dissociate rapidly for the cell to detect the lesion and mount a DNA damage response. Understanding how these two critical steps are modulated by the target sequence will help researchers optimize target site selection and guide RNA design. Chromatin remodelers are ATPases that changes the position or composition of nucleosomes through nucleosome sliding or histone exchange, respectively. Many pioneering studies, including those using single- molecule methods, have revealed the fine details of the sliding reaction, but our knowledge of the histone exchange reaction is much more limited. We propose to develop single-molecule assays to fully define the kinetics and pathways of SWR1-catalyzed exchange of H2A-H2B dimer for H2A.Z-H2B dimer. We will also discover and characterize yeast native genomic sequences that allow nucleosome formation at a sharply defined location, which we hope will replace the artificial nucleosome positioning sequence, `601', that has been used in almost all mechanistic studies involving nucleosomes. DNA mismatch repair is carried out by conserved protein machinery but despite the decades of research, we have a very limited understanding of how different mismatches under different sequence contexts are repaired. For example, most mechanistic studies have been performed on a single type of mismatch (GT mismatch). We will develop a high throughput method to measure the in vivo mismatch repair efficiencies of thousands of different mismatches. If unrepaired until histone deposition on a nascent DNA, a mismatch will become part of a nucleosome. We will study the biophysical properties of a mismatch-containing nucleosome and its interplay with chromatin remodelers, allowing us to examine how an unrepaired mismatch can influence DNA accessibility.