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DNA复制和修复所必需的许多酶中都发现了铁-硫簇。这里 我们提出了这些至关重要的DNA加工的氧化还原化学的特征 酶以及如何利用这种化学作用通过DNA电荷传输进行远程信号传递 (CT)跨越基因组。我们将使用DNA电化学来表征DNA结合电位和 DNA加工蛋白包括DNA聚合酶α、聚合酶δ和 Primase以及几种DNA修复蛋白。实验将在厌氧条件下进行，使用 多路DNA芯片。氧化的蛋白质和还原的蛋白质将通过电化学的方法制备，DNA和 含[4Fe4S]簇的蛋白质在2+VS 3+中的结合和蛋白质活性将被确定 表格。在进行DNA CT的熟练程度方面存在差异的突变蛋白质也将 分离和表征电化学和生物化学，包括人类蛋白质的突变 已知会促进结肠癌。一旦氧化和还原的酶信号已经被 建立后，我们将通过电化学或结合光氧化剂来制备氧化蛋白质， 并使用氧化蛋白作为另一种DNA结合蛋白的信号伙伴。检测至 确定氧化信号将包括复制活性的测量和AFM分析 用单碱基测定精通CT的[4Fe4S]蛋白在DNA链上的定位 不匹配。CT缺陷突变体将被并行检查，提供关键控制以及 描述与这些突变相关的癌变的手段。这些实验 为细胞内远距离DNA介导的信号传递提供体外模型。我们还将检查 含有[4Fe4S]的DNA修复蛋白在大肠杆菌中的远程信号传递 集群。我们将利用几种体内修复试验来检查如何从基因上敲除一个 蛋白质影响另一种蛋白质的活性。这些基因实验应该可以确定 不同修复途径之间可能存在的DNA信号网络。与质粒互补 CT缺陷型和熟练型突变体都将被检测。我们还将设计类似的 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。我们还将检查是否有其他涉及DNA的大肠杆菌蛋白质- 处理包含[4Fe4S]簇并参与DNA信号传递，包括UvrC和 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 跨基因组的信号和疾病中DNA CT缺陷的重要考虑。