NMR studies of a model DNA polymerase

模型 DNA 聚合酶的 NMR 研究

基本信息

批准号：
8911836
负责人：
Zucai Suo
金额：
$ 22.26万
依托单位：
OHIO STATE UNIVERSITY
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：
2014-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8911836
关键词：
Adopted Apoptosis Arts Binding Biological Process Bypass Catalysis Cells Complex DNA DNA Binding DNA Damage DNA Polymerase beta DNA Repair DNA Sequence DNA biosynthesis DNA lesion DNA-Directed DNA Polymerase Enzymes Family Fingers Fluorescence Resonance Energy Transfer Foundations Future Generations Genomic DNA Genomics Goals Immunoglobulins Kinetics Label Literature Measurement Modeling Molecular Molecular Conformation Motion Nature Nucleotides Organism PAWR protein Pathway interactions Phase Play Polymerase Property Proteins Publishing Roentgen Rays Role Sampling Scheme Signal Transduction Single-Stranded DNA Sister Chromatid Site Solutions Structure Sulfolobus solfataricus Techniques Thumb structure Time Uncertainty Work base cohesion in vivo insight instrumentation phosphodiester polymerization preference protein function protein structure protonation public health relevance stopped-flow fluorescence

项目摘要

DESCRIPTION (provided by applicant): DNA polymerases perform a diverse repertoire of biological functions including genomic replication, DNA damage repair, DNA lesion bypass, immunoglobulin generation, and sister chromatid cohesion. These enzymes are phylogenetically grouped into six families (A, B, C, D, X, and Y). DNA polymerases from different families differ in their in vivo functions, primary sequences, DNA substrate preferences (non-gapped, gapped, or single-stranded DNA; damaged or undamaged DNA), nucleotide incorporation efficiency and fidelity, and polymerization processivity. Mechanistically, all kinetically characterized DNA polymerases share a minimal kinetic mechanism of nucleotide incorporation including several hypothetical protein conformational changes, one of them being rate-limiting. Although X-ray crystallographic studies of DNA polymerases have suggested that the rate-limiting conformational change involves a dramatic closing of the Finger domain alone, several recent stopped-flow fluorescence resonance energy transfer (FRET) studies of several DNA polymerases including Dpo4 indicate that the Finger domain closing is too fast to limit correct nucleotide incorporation. Dpo4 is from Sulfolobus solfataricus and belongs to the Y-family polymerases, which can bypass DNA lesions, thereby rescuing cells from apoptosis. The long term goal of the PIs is to understand how the structures and conformational dynamics of a DNA polymerase play a role in its enzymatic function. Recent stopped-flow FRET studies of Dpo4 by the Contact PI's group have revealed that all four structural domains of Dpo4, not just the Finger domain, undergo rapid conformational changes before and after phosphodiester bond formation. These observed motions were unfortunately invisible from numerous published X-ray crystallographic studies. More interestingly, when a DNA lesion is encountered, several domains of Dpo4 adopt different conformations as compared to those observed with a normal DNA substrate. To understand the exact nature of these domain motions in solution and their connection with the kinetic mechanism of nucleotide incorporation, we will employ solution NMR techniques to investigate this hyperthermostable and relatively small sized DNA polymerase, Dpo4 , with the following two specific aims: 1) Determine the solution structures of apo Dpo4 and its binary (Dpo4??DNA) and ternary (Dpo4??DNA??dNTP) complexes; 2) Investigate conformational dynamics of Dpo4 in its apo form, binary (Dpo4??DNA) and ternary (Dpo4??DNA??dNTP) complexes. Once completed, our proposed studies will reveal detailed site-specific information on changes in protein structure and/or conformational dynamics that are important for substrate (DNA, correct or incorrect nucleotide) binding and catalysis. More importantly, our studies will unravel any structural and dynamic determinants responsible for the fidelity of a Y-family DNA polymerase during translesion DNA synthesis.

描述（由申请人提供）：DNA聚合酶执行多种生物学功能，包括基因组复制、DNA损伤修复、DNA损伤旁路、免疫球蛋白生成和姐妹染色单体凝聚。这些酶在系统发育上分为六个家族（A、B、C、D、X 和 Y）。不同家族的 DNA 聚合酶在体内功能、一级序列、DNA 底物偏好（无缺口、缺口或单链 DNA；受损或未受损 DNA）、核苷酸掺入效率和保真度以及聚合持续性方面有所不同。从机制上讲，所有动力学特征的 DNA 聚合酶都共享核苷酸掺入的最小动力学机制，包括几种假设的蛋白质构象变化，其中之一是限速的。尽管 DNA 聚合酶的 X 射线晶体学研究表明，限速构象变化仅涉及 Finger 结构域的急剧关闭，但最近对包括 Dpo4 在内的几种 DNA 聚合酶进行的几项停流荧光共振能量转移 (FRET) 研究表明，指状结构域关闭太快，无法限制正确的核苷酸掺入。 Dpo4来自硫磺硫化叶菌（Sulfolobus solfataricus），属于Y家族聚合酶，可以绕过DNA损伤，从而拯救细胞免于凋亡。 PI 的长期目标是了解 DNA 聚合酶的结构和构象动力学如何在其酶功能中发挥作用。 Contact PI 小组最近对 Dpo4 进行的停流 FRET 研究表明，Dpo4 的所有四个结构域（不仅仅是 Finger 结构域）在磷酸二酯键形成之前和之后都会经历快速的构象变化。不幸的是，这些观察到的运动在许多已发表的 X 射线晶体学研究中是看不见的。更有趣的是，当遇到 DNA 损伤时，Dpo4 的几个结构域会采用与正常 DNA 底物观察到的结构不同的构象。为了了解溶液中这些结构域运动的确切性质及其与核苷酸掺入动力学机制的联系，我们将采用溶液 NMR 技术来研究这种超热稳定且相对较小尺寸的 DNA 聚合酶 Dpo4，具有以下两个具体目标：1)确定apo Dpo4及其二元(Dpo4??DNA)和三元(Dpo4??DNA??dNTP)复合物的溶液结构； 2）研究Dpo4的apo形式、二元（Dpo4??DNA）和三元（Dpo4??DNA??dNTP）复合物的构象动力学。一旦完成，我们提出的研究将揭示有关蛋白质结构和/或构象动力学变化的详细位点特定信息，这些信息对于底物（DNA、正确或不正确的核苷酸）结合和催化非常重要。更重要的是，我们的研究将揭示在跨损伤 DNA 合成过程中负责 Y 家族 DNA 聚合酶保真度的任何结构和动态决定因素。