Protein Dynamics in Site-Specific DNA Recombination

位点特异性 DNA 重组中的蛋白质动力学

基本信息

批准号：
9883005
负责人：
MARK P. FOSTER
金额：
$ 10.49万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9883005
关键词：
Address Adopted Affect Animal Model Binding Biotechnology Cell division Cell physiology Cells Chromosome Pairing Cicatrix Communication Complex Cruciform DNA Crystallization DNA DNA Binding DNA Sequence Engineering Enterobacteria phage P1 Cre recombinase Enzymes Excision Free Energy Genes Genetic Engineering Genetic Recombination Geometry Goals Health Homo Human Integration Host Factors Isomerism Isotope Labeling Knowledge Label Left Ligation Maps Measurement Measures Mediating Methods Molecular Conformation NMR Spectroscopy Pathogenesis Pathway interactions Phosphotyrosine Process Protein Dynamics Proteins Protomer Reaction Reagent Relaxation Resolution Role Series Site Spacer DNA Specificity Structure Synapses Techniques Technology Therapeutic Tyrosine Variant Virus Diseases Work Zonula Adherens base cofactor design dimer experimental study flexibility genome editing improved innovation insight recombinase repaired restriction enzyme structured data tool

项目摘要

Project Summary/Abstract This goal of this proposal is to advance our understanding of the mechanisms by which tyrosine recombinases recognize and assemble on their target DNA sequences and achieve coordinated pairwise cleavage of DNA strands to reach a recombined product. The proposal is significant because tyrosine recombinases are widely used genome editing tools, but their potential for improving human health is currently hindered by lack of understanding of their mechanisms for site selection and for control over activity and recombination direction (i.e., integration versus excision). Innovation in this proposal arises from the application of solution NMR to address mechanistic knowledge gaps left unanswered by the many high resolution crystal structures of tyrosine recombinases in tetrameric complexes with DNA. Those structures have provided crisp snapshots of some of the important intermediates in the recombination pathway, but provide limited insight into the intermediates that precede them, or into the mechanisms that interconvert them. Our approach combines powerful protein- and DNA-engineering with sophisticated isotope labeling and NMR methods to characterize dynamics that enable interconversion of key intermediates in site-specific DNA recombination, focusing on those leading to site-specific assembly of the tetrameric synaptic complex, allosteric control over DNA cleavage, and isomerization of the Holliday junction (HJ) intermediate. To understand the conformational changes in both protein and DNA that accompany site selection, dimer assembly, tetramer synapsis and protomer activation, we will use solution NMR spectroscopy to: (1) determine the solution structure of Cre recombinase alone, and bound in pre-synapsed complexes with loxP DNA; (2) determine the role of protein dynamics in activating Cre for DNA cleavage by measuring dynamics in Cre and pre-synaptic complexes with DNA; (3) study how DNA intrinsic dynamics affects Cre recognition, synaptic assembly, control over Cre activity, and direction of recombination; (4) characterize the dynamic and allosteric communication pathways that enable isomerization of the central HJ for progression through the recombination reaction. To enable the NMR experiments on large homo-oligomeric complexes, we will leverage an arsenal of reagents and techniques for selective labeling of protein and DNA molecules, and for assembly of chimeric Cre-DNA complexes; together with uniform deuteration and TROSY methods, the simplified NMR spectra will facilitate resonance assignments and quantitative relaxation measurements. The proposed studies will advance our understanding of the role of dynamics in DNA recombination, and in DNA binding and remodeling enzymes in general. This knowledge could broadly impact biotechnology and its biomedical applications by facilitating the design of Cre variants with defined DNA sequence specificity and improved efficiency, and suggest new avenues for controlling its activity.

项目概要/摘要该提案的目标是增进我们对酪氨酸重组酶机制的理解识别并组装其目标 DNA 序列并实现 DNA 的协调成对切割链以达到重组产物。该提案意义重大，因为酪氨酸重组酶被广泛应用使用基因组编辑工具，但其改善人类健康的潜力目前因缺乏了解其位点选择机制以及控制活性和重组方向的机制（即整合与切除）。该提案的创新源于溶液核磁共振的应用解决许多高分辨率晶体结构留下的机械知识空白酪氨酸重组酶与 DNA 形成四聚体复合物。这些结构提供了清晰的快照重组途径中的一些重要中间体，但对重组途径的了解有限它们之前的中间体，或进入它们相互转化的机制。我们的方法结合了强大的蛋白质和 DNA 工程，具有复杂的同位素标记和 NMR 方法来表征能够实现位点特异性 DNA 重组中关键中间体相互转化的动力学，重点关注那些导致四聚体突触复合体的位点特异性组装、DNA变构控制霍利迪连接体 (HJ) 中间体的裂解和异构化。为了了解蛋白质和 DNA 中伴随位点选择的构象变化，二聚体组装、四聚体突触和原聚体激活，我们将使用溶液核磁共振波谱来：（1）确定单独的 Cre 重组酶的溶液结构，并与 loxP DNA 结合在突触前复合物中； (2) 通过测量 Cre 和 DNA 裂解中的动力学，确定蛋白质动力学在激活 Cre 中的作用与 DNA 的突触前复合物； (3)研究DNA内在动力学如何影响Cre识别、突触组装、Cre 活性控制和重组方向； (4) 表征动态和变构使中央 HJ 异构化并通过重组进行进展的通讯途径反应。为了能够对大型同源寡聚复合物进行 NMR 实验，我们将利用一系列用于选择性标记蛋白质和 DNA 分子以及用于组装嵌合体的试剂和技术 Cre-DNA 复合物；与均匀氘化和 TROSY 方法一起，简化的 NMR 谱将促进共振分配和定量弛豫测量。拟议的研究将增进我们对 DNA 重组、DNA 结合和重塑中动力学作用的理解一般而言，酶。这些知识可能会广泛影响生物技术及其生物医学应用促进具有明确 DNA 序列特异性和更高效率的 Cre 变体的设计，以及提出控制其活动的新途径。