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

• 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
• 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

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）：解旋酶包括一大组酶，从“经典”到“假解旋酶”，其在基因组维持中起重要作用。假解旋酶在从癌症、染色质重塑到远距离DNA位点之间的远距离通讯等生物学过程中一直是研究热点。虽然一些假解旋酶是消耗数百个ATP分子以在DNA/RNA上进行前向移动的真正的马达或移位酶，但其他的被证明是“分子开关”，其仅水解几个ATP以切换结构状态，从而使DNA/RNA的结构状态发生改变。 长距离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非常重要，但它们的作用机制仍然是个谜。 III型限制性内切酶（RE）提供了理想的系统来抑制假解旋酶活性，因为所有的酶功能都整合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组实验相结合的X射线晶体学与国家的最先进的单分子和合奏测量，以阐明如何EcoP 15 I，III型RE家族的原型，从一个状态过渡到另一个长寿命的滑动DNA。 目的一：首次获得EcoP 15 Ⅰ的三维结构信息。 除了天然Ecop 15 I/DNA复合物，我们将确定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III型限制性内切酶，只有第二个解旋酶绑定到双链DNA。在目标2中，我们将推导出 解释结构结果。 在结构的指导下，我们将进行单分子和整体荧光共振能量转移测量EcoP 15 I与DNA和ATP相互作用过程中的动力学。 我们将利用一个专门建造的磁性镊子全内反射荧光（MT-TIRF）显微镜，可以可视化单个荧光标记的蛋白质滑动沿着DNA在磁场中拉伸。 这将通过使用停止流动的毫秒时间分辨率荧光测定以及新的基于Biacore的DNA解离测定来补充。 总之，拟议的“实时”分析将补充结构研究，并提供前所未有的新细节的反应途径的ATP依赖性分子开关在DNA代谢。