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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聚合酶具有多种生物学功能，包括基因组复制、DNA损伤修复、DNA损伤绕道、免疫球蛋白生成和姐妹染色单体内聚。这些酶在系统发育上分为6个家族（A、B、C、D、X和Y）。来自不同家族的DNA聚合酶在体内功能、初级序列、DNA底物偏好（非缺口、缺口或单链DNA；受损或未受损的DNA）、核苷酸结合效率和保真度以及聚合能力方面存在差异。从机制上讲，所有具有动力学特征的DNA聚合酶都具有核苷酸结合的最小动力学机制，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结构域的剧烈关闭，但最近对包括Dpo4在内的几种DNA聚合酶的停流荧光共振能量转移（FRET）研究表明，Finger结构域的关闭速度太快，无法限制正确的核苷酸整合。Dpo4来自于solfataricus Sulfolobus，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。最近，Contact PI的团队对Dpo4进行了停止流动的FRET研究，揭示了Dpo4的所有四个结构域，而不仅仅是Finger结构域，在磷酸二酯键形成前后都经历了快速的构象变化。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，与正常DNA底物相比，Dpo4的几个结构域采用不同的构象。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结合动力学机制的联系，我们将采用溶液核磁共振技术来研究这种超热稳定性和相对小尺寸的DNA聚合酶Dpo4，有以下两个具体目的：1)确定载子Dpo4及其二元(Dpo4?？DNA)和三元(Dpo4??DNA？核苷酸)复合物;2)研究载子形式Dpo4的构象动力学，二元（Dpo4??）DNA)和三元(Dpo4??DNA？核苷酸)复合物。一旦完成，我们提出的研究将揭示蛋白质结构和/或构象动力学变化的详细位点特异性信息，这些信息对底物（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翻译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。