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 铁硫簇存在于许多DNA复制和修复所必需的酶中。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， 酶以及如何通过DNA电荷传输将这种化学物质用于长距离信号传导 (CT)在基因组中。我们将使用DNA电化学来表征DNA结合电位， DNA加工蛋白包括DNA聚合酶α、聚合酶δ和 引物酶以及几种DNA修复蛋白。实验将在厌氧条件下进行， 多重DNA芯片氧化蛋白质与还原蛋白质将通过电化学方法制备， 结合和蛋白质活性将被确定为蛋白质与[4Fe 4S]簇在2+ vs 3+ forms.在进行DNA CT的能力方面不同的突变蛋白质也将被 分离和表征电化学和生物化学，包括人类蛋白质的突变 会导致结肠癌一旦氧化与还原的酶特征被确定， 建立，我们将制备氧化蛋白质，无论是电化学或与拴光氧化剂， 并使用氧化蛋白作为另一种DNA结合蛋白的信号伴侣。测定以 确定氧化信号传导将包括复制活性的测量和AFM测定， 测量CT-精通[4Fe 4S]蛋白在DNA链上的定位， 不匹配. CT缺陷突变体将平行检查，提供关键对照以及 是指描述与这些突变相关的癌性转化。这些实验 为细胞内的长距离DNA介导的信号传导提供体外模型。我们亦会研究 大肠杆菌中含有[4Fe 4S]的DNA修复蛋白之间的长程信号传导 集群我们将利用几种体内修复试验来研究基因敲除一个 一种蛋白质影响另一种蛋白质的活性。这些遗传实验应该可以确定 不同修复途径之间的DNA信号传导的可能网络。与质粒互补 将检查CT缺陷型和CT精通型突变体。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节蛋白质拷贝数， 探索对DNA信号的影响。我们还将研究其他E。大肠杆菌蛋白质参与DNA- 处理含有[4Fe 4S]簇并参与DNA信号传导，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 跨基因组的信号传导和疾病中DNA CT缺陷的重要考虑。