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

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

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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.

项目摘要/摘要该提议的这个目标是提高我们对酪氨酸重组酶的机制的理解识别并组装其目标DNA序列并实现DNA的配对裂解链以达到重组产品。该提案很重要，因为酪氨酸重组酶广泛使用的基因组编辑工具，但由于缺乏了解他们的现场选择机制以及控制活动和重组方向的控制（即集成与切除）。该提案中的创新是由解决方案NMR应用于解决机械知识的差距，没有由许多高分辨率晶体结构所搁置酪氨酸重组酶与DNA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，但对中间在它们之前或互换的机制中进行了中间。我们的方法结合在一起具有复杂的同位素标签和NMR方法的强大蛋白质和DNA工程来表征能够在特定地点DNA重组中互转换的动力学，重点是那些导致四聚体突触复合物，对DNA的变构控制的特定地点组装裂解和霍利迪交界处（HJ）中级的异构化。了解伴随位点选择的蛋白质和DNA的构象变化，二聚体组装，四聚体突触和原始物激活，我们将使用溶液NMR光谱法进行：（1）确定仅CRE重组酶的溶液结构，并与LOXP DNA的预共同复合物结合；（2）通过测量CRE和与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，控制CRE活性和重组方向；（4）表征动态和变构的使中央HJ通过重组进展的中央HJ的沟通途径反应。为了实现大型同源物种复合物的NMR实验，我们将利用选择性标记蛋白质和DNA分子的试剂和技术，以及用于嵌合的组装 Cre-DNA复合物；与统一的剥离和trosy方法一起，简化的NMR光谱将促进共振分配和定量放松测量。拟议的研究将促进我们对动力学在DNA重组以及DNA结合和重塑中的作用的理解酶通常。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定义的DNA序列特异性和提高效率的CRE变体的设计，以及提出了控制其活动的新途径。