DNA Processing Enzymes with [4Fe4S] Clusters for DNA Signaling

用于 DNA 信号转导的具有 [4Fe4S] 簇的 DNA 加工酶

• 批准号: 9146616
• 负责人: JACQUELINE K BARTON
• 金额: $ 29.94万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9146616
• 关键词: Active Biological Transport; Affect; Base Excision Repairs; Base-Base Mismatch; Biochemical; Biological Assay; CRISPR interference; CRISPR/Cas technology; Cancerous; Cells; Charge; Chemistry; Colon Carcinoma; DNA; DNA Binding; DNA Microarray Chip; DNA Primase; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Disease; Electrochemistry; Engineering; Enzymes; Escherichia coli; Escherichia coli Proteins; Excision; Genetic; Genome; Genomics; Growth; Human; Investigation; Iron; Knock-out; Malignant Neoplasms; Measurement; Measures; Mediating; Modeling; Monitor; Mutation; Nucleotides; Oxidation-Reduction; Pathway interactions; Plasmids; Polymerase; Process; Proteins; Role; Signal Transduction; Sulfur; Type I DNA Topoisomerases; Work; endonuclease III; enzyme activity; genome editing; in vitro Model; in vitro activity; in vivo; mutant; oxidation; repair enzyme; repaired; research study; ultraviolet irradiation

Description Iron-sulfur clusters have been found in many enzymes essential for DNA replication and repair. Here we propose the characterization of the redox chemistry of these critically important DNA-processing enzymes and how this chemistry may be utilized for long range signaling through DNA charge transport (CT) across the genome. We will use DNA electrochemistry to characterize DNA-bound potentials and DNA CT proficiencies of DNA-processing proteins including DNA polymerase α, polymerase δ and primase as well as several DNA repair proteins. Experiments will be conducted anaerobically using multiplexed DNA chips. Oxidized vs reduced proteins will be prepared electrochemically and both DNA binding and protein activities will be determined for the proteins with [4Fe4S] clusters in 2+ vs 3+ forms. Mutant proteins that differ with respect to their proficiency in carrying out DNA CT will also be isolated and characterized electrochemically and biochemically, including mutations in human proteins known to promote colon cancer. Once the enzyme signatures for oxidation vs reduction have been established, we will prepare oxidized proteins, either electrochemically or with tethered photooxidants, and use the oxidized protein as a signaling partner for another DNA-bound protein. Assays to determine oxidative signaling will include measurements of replication activity and an AFM assay to measure the localization of CT-proficient [4Fe4S] proteins onto DNA strands with a single base-base mismatch. CT-deficient mutants will be examined in parallel, providing critical controls as well as a means to describe cancerous transformations associated with these mutations. These experiments provide an in vitro model for long-range DNA-mediated signaling within the cell. We will also examine long range-range signaling within Escherichia coli among DNA repair proteins that contain [4Fe4S] clusters. We will utilize several in vivo assays for repair to examine how genetically knocking out one protein affects the activity of another. These genetic experiments should allow the determination of possible networks for DNA signaling among different repair pathways. Complementation with plasmids of both CT-deficient and -proficient mutants will be examined. We will also engineer analogous genomic mutations using CRISPR/Cas9, and CRISPRi will be used to tune protein copy number and explore effects on DNA signaling. We will also examine whether other E. coli proteins involved in DNA- processing contain [4Fe4S] clusters and participate in DNA signaling, including UvrC and topoisomerase I. This work will contribute both to our fundamental understanding of protein/DNA signaling across the genome and the important consideration of DNA CT deficiencies in disease.

描述 在许多酶中发现了铁硫簇，对于DNA复制和修复必不可少的酶。这里 我们提出了这些至关重要的DNA处理的氧化还原化学的表征 酶以及如何通过DNA电荷传输将这种化学用于远程信号传导 （CT）整个基因组。我们将使用DNA电化学来表征DNA结合电位，并且 DNA CT的DNA处理蛋白包括DNA聚合酶α，聚合酶δ和 原始酶以及几种DNA修复蛋白。实验将在厌氧上使用 多路复用DNA芯片。氧化与还原蛋白的蛋白质将通过电化学制备，并且均为DNA 将确定具有[4FE4]簇的蛋白质的结合和蛋白质活性 表格。突变蛋白在执行DNA CT方面不同的突变蛋白也将是 分离和表征的电化学和生化，包括人蛋白的突变 已知可以促进结肠癌。一旦氧化的酶特征与还原相对于还原 建立，我们将在电化学或用束缚的光氧化剂上制备氧化物蛋白 并使用氧化蛋白作为另一个DNA结合蛋白的信号伴侣。测定 确定氧化信号传导将包括测量复制活动和AFM测定 测量具有单个基碱基的CT促进[4FE4S]蛋白在DNA链上的定位 不匹配。 CT缺陷突变体将并行检查，提供关键控制和 描述与这些突变相关的取消转换的手段。这些实验 为细胞内的远程DNA介导的信号传导提供体外​​模型。我们还将检查 含有[4FE4S]的DNA修复蛋白中大肠杆菌内的远程信号传导 集群。我们将利用几种体内测定进行维修，以检查如何一般敲出一个 蛋白质会影响另一种的活性。这些遗传实验应允许确定 不同修复途径之间的DNA信号的可能网络。与浆质的互补 将检查CT缺陷型和优先突变体的中。我们还将设计类似 使用CRISPR/CAS9和CRISPRI的基因组突变将用于调整蛋白质拷贝数和 探索对DNA信号的影响。我们还将检查其他参与DNA-的大肠杆菌蛋白是否 处理包含[4FE4]簇并参与包括UVRC和包括UVRC的DNA信号传导的处理 拓扑异构酶I。这项工作将有助于我们对蛋白质/DNA的基本理解 跨基因组的信号传导以及疾病中DNA CT缺陷的重要考虑。