Structure-function analysis of a molecular switch for long-range diffusion on DNA

DNA 长程扩散分子开关的结构功能分析

基本信息

批准号：
8927038
负责人：
ANEEL K. AGGARWAL
金额：
$ 31.57万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-15 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8927038
关键词：
3-Dimensional ATP Hydrolysis ATP phosphohydrolase Amino Acid Motifs Articular Range of Motion Back Bacterial Typing Base Pairing Binding Biochemical Biological Assay Biological Process Characteristics Collaborations Communication Complement Complex Coupled Cysteine DNA DNA Binding DNA Repair DNA Restriction Enzymes DNA Sequence Diffusion Dissociation Distant Dyes Employee Strikes Engineering Enzymes Event Family Fluorescence Resonance Energy Transfer Genome Germany Goals Health Holoenzymes Hydrolysis International Investigation Kinetics Label Life Magnetism Maintenance Malignant Neoplasms Measurement Metabolism Methylation Mismatch Repair Modification Molecular Motion Motor Multienzyme Complexes Names Nucleoproteins Nucleotide Excision Repair Nucleotides Pathway interactions Play Polynucleotides Positioning Attribute Process Proteins RNA Reaction Resolution Roentgen Rays Role Shapes Signal Transduction Site Slide Stretching Structure Synapses System Testing Time Universities X-Ray Crystallography analog base biophysical analysis biophysical techniques bone chromatin remodeling cofactor conformational conversion ds-DNA enzyme mechanism enzyme structure fluorescence microscope helicase innovation insight magnetic field medical schools millisecond nuclease protein protein interaction prototype research study restriction enzyme single molecule structural biology translocase

项目摘要

DESCRIPTION (provided by applicant): Helicases comprise a large group of enzymes, from "classical" to "pseudo-helicases", which play important roles in genome maintenance. The pseudo-helicases have been the subject intense investigation in biological processes ranging from cancer, chromatin remodeling, to long-range communication between distant DNA sites. While some of pseudo-helicases are bone-fide motors or translocases that consume hundreds of ATP molecules to processively move on the DNA/RNA, others are turning out to be "molecular switches" that hydrolyze just a few ATPs to switch structural states for long-range diffusion. These molecular switches are important in processes ranging from nucleotide excision repair to mismatch repair, but their mechanism of action remains mysterious. The Type III restriction enzymes (REs) offer the ideal system to investigat pseudo-helicase activity because all of the enzymatic functions are integrated in the same holoenzyme complex and no additional protein cofactors are required. We propose here a set of experiments combining X-ray crystallography with state-of-the-art single-molecule and ensemble measurements to elucidate how, EcoP15I, a prototype of the Type III RE family, transitions from one state to another for long-lived sliding on DNA. In Aim 1, we wil derive the first 3-D structural information on EcoP15I. In addition to the native Ecop15I/DNA complex, we will determine structures in the presence of ADP and ATP analogues, as well as structure of the enzyme in synaptic or "collision" complex. The proposed structural studies are the first for a Type III restriction enzyme, and only the second for a helicase bound to double-stranded DNA. In aim 2, we will derive a kinetic framework for the interpretation of structural results. Guided by the structure, we will perform single molecule and ensemble fluorescence resonance energy transfer measurements of EcoP15I dynamics during interaction with DNA and ATP. We will take advantage of a specially built magnetic tweezers-total internal reflection fluorescence (MT-TIRF) microscope that can visualize single fluorescently- labeled proteins sliding along DNA stretched within a magnetic field. This will be complemented by millisecond time resolution fluorescent assays using stopped flow, as well as new Biacore-based DNA dissociation assays. Together, the proposed "real-time" assays will complement the structural studies and provide unprecedented new details on the reaction pathway of an ATP-dependent molecular switch in DNA metabolism.

描述（由申请人提供）：解旋酶包含一大类酶，从“经典”到“伪解旋酶”，它们在基因组维护中发挥重要作用。假螺旋酶一直是从癌症、染色质重塑到遥远 DNA 位点之间的长距离通讯等生物过程中广泛研究的主题。虽然一些伪解旋酶是真正的马达或易位酶，它们消耗数百个 ATP 分子以在 DNA/RNA 上持续移动，但其他伪解旋酶被证明是“分子开关”，仅水解几个 ATP 即可切换结构状态长程扩散。这些分子开关在从核苷酸切除修复到错配修复的过程中都很重要，但它们的作用机制仍然神秘。 III 型限制性内切酶 (RE) 提供了研究假解旋酶活性的理想系统，因为所有酶功能都集成在同一个全酶复合物中，并且不需要额外的蛋白质辅因子。我们在这里提出了一系列实验，将 X 射线晶体学与最先进的单分子和整体测量相结合，以阐明 EcoP15I（III 型 RE 系列的原型）如何长期从一种状态转变为另一种状态。靠 DNA 生活。在目标 1 中，我们将获得 EcoP15I 的第一个 3D 结构信息。除了天然的 Ecop15I/DNA 复合物之外，我们还将确定 ADP 和 ATP 类似物存在下的结构，以及突触或“碰撞”复合物中酶的结构。拟议的结构研究是第一个针对 III 型限制性内切酶的结构研究，也是第二个针对与双链 DNA 结合的解旋酶的结构研究。在目标 2 中，我们将为结构结果的解释。在该结构的指导下，我们将对 EcoP15I 与 DNA 和 ATP 相互作用过程中的动力学进行单分子和整体荧光共振能量转移测量。我们将利用特制的磁性镊子全内反射荧光 (MT-TIRF) 显微镜，该显微镜可以可视化沿着磁场内拉伸的 DNA 滑动的单个荧光标记蛋白质。这将得到使用停流的毫秒时间分辨率荧光测定以及基于 Biacore 的新 DNA 解离测定的补充。总之，所提出的“实时”测定将补充结构研究，并提供关于 DNA 代谢中 ATP 依赖性分子开关的反应途径的前所未有的新细节。