Protein Dynamics in Site-Specific DNA Recombination

项目总结/摘要 这个建议的目的是为了促进我们对酪氨酸重组酶的机制的理解， 识别并组装在其靶DNA序列上，并实现DNA的协调成对切割 以获得重组产品。该提议是重要的，因为酪氨酸重组酶广泛存在于 使用基因组编辑工具，但由于缺乏技术，它们改善人类健康的潜力目前受到阻碍 了解它们的位点选择机制以及对活性和重组方向的控制机制 (i.e.,整合与切除）。该建议的创新源于溶液NMR的应用， 解决机械知识空白留下的许多高分辨率的晶体结构， 与DNA形成四聚体复合物的酪氨酸重组酶。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，但提供了有限的见解， 中间体之前，或进入相互转换它们的机制。我们的方法结合了 强大的蛋白质和DNA工程与复杂的同位素标记和NMR方法来表征 动力学，使位点特异性DNA重组中的关键中间体相互转化，重点是 那些导致四聚体突触复合物的位点特异性组装，对DNA的变构控制 裂解和霍利迪结（HJ）中间体的异构化。 为了理解伴随位点选择的蛋白质和DNA的构象变化， 组装，四聚体突触和原聚体活化，我们将使用溶液NMR光谱：（1）确定 Cre重组酶单独的溶液结构，并与loxP DNA结合在突触前复合物中;（2） 通过测量Cre中的动力学来确定蛋白质动力学在激活Cre以进行DNA切割中的作用， （3）研究DNA内在动力学如何影响Cre识别、突触前复合物和DNA的相互作用。 组装，控制Cre活性和重组方向;（4）表征动态和变构 通讯途径，使异构化的中央HJ的进展，通过重组 反应为了能够在大的同源寡聚复合物上进行NMR实验，我们将利用 用于蛋白质和DNA分子的选择性标记以及用于嵌合体组装的试剂和技术 Cre-DNA复合物;连同均匀氘化和TROSY方法，简化的NMR谱将 便于共振分配和定量弛豫测量。拟议的研究将 推进我们对动力学在DNA重组、DNA结合和重塑中的作用的理解 一般的酵素这些知识可以广泛影响生物技术及其生物医学应用， 促进具有确定的DNA序列特异性和提高的效率的Cre变体的设计，和 提出了控制其活动的新途径。

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.