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

基本信息

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
项目类别：
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

项目摘要

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 DNA 复制和修复所必需的酶中都发现了铁硫簇。我们提出了这些极其重要的 DNA 处理的氧化还原化学的表征酶以及如何利用这种化学物质通过 DNA 电荷传输进行长距离信号传导我们将使用 DNA 电化学来表征 DNA 结合电位和 DNA CT 熟练掌握 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 修复蛋白将在厌氧条件下进行。多重 DNA 芯片将通过电化学方法制备，并同时制备两种 DNA。将确定 2+ 与 3+ 中具有 [4Fe4S] 簇的蛋白质的结合和蛋白质活性其执行 DNA CT 的能力也有所不同的突变蛋白。通过电化学和生物化学方法进行分离和表征，包括人类蛋白质的突变一旦氧化与还原的酶特征被确定，就会促进结肠癌。建立后，我们将通过电化学或束缚光氧化剂制备氧化蛋白质，并使用氧化蛋白作为另一种 DNA 结合蛋白的信号传导伴侣进行检测。确定氧化信号将包括复制活性的测量和 AFM 测定使用单碱基测量 CT 熟练的 [4Fe4S] 蛋白在 DNA 链上的定位 CT 缺陷突变体将被并行检查，提供关键对照和意味着描述与这些突变相关的癌变。我们还将研究细胞内长程 DNA 介导的信号传导的体外模型。大肠杆菌内含有 [4Fe4S] 的 DNA 修复蛋白之间的长距离信号传导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簇。这些基因实验应该可以确定蛋白质的活性。不同修复途径之间 DNA 信号传导的可能网络。我们还将检查 CT 缺陷型和 CT 型突变体的相似性。使用 CRISPR/Cas9 进行基因组突变，CRISPRi 将用于调整蛋白质拷贝数和我们还将研究其他大肠杆菌蛋白是否参与 DNA 信号传导。加工包含[4Fe4S]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以及疾病中 DNA CT 缺陷的重要考虑。