ERROR CORRECTION IN DNA SYNTHESIS--A BIOCHEMICAL STUDY

DNA 合成中的错误纠正——一项生化研究

• 批准号: 6618541
• 负责人: MYRON GOODMAN
• 金额: $ 10万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6618541
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; Schizosaccharomyces pombe; active sites; bacterial proteins; chemical kinetics; enzyme activity; exonuclease; fluorescent dye /probe; fungal proteins; gene mutation; hydrogen bond; nucleic acid sequence; nucleotide analog

The broad objective of this grant proposal is to study the mechanisms responsible for the fidelity of DNA synthesis. DNA polymerases are the key enzymes involved in replication and repair of DNA. An analysis of how polymerases control fidelity is central to understanding the biochemical basis of a wide variety of genetic diseases. Lesch-Nyhan syndrome and ADA deficiency are two examples of inherited childhood diseases that can arise from a single point mutation. Activation of oncogenes and inactivation of tumor suppressor genes leading to cancer can result from single base changes in DNA. Genetic defects in post-replication mismatch repair of DNA polymerase errors are a root cause of hereditary nonpolyposis colin cancer, along with a variety of other types of cancer. Previous fidelity studies have focused on individual DNA polymerases in the absence of polymerase accessory proteins required to sustain processive synthesis. This grant investigates the fidelity of purified procaryotic and eucaryotic DNA polymerase holoenzymes, pol III and pol II from Escherichia coli, and pol delta from Schizosaccharomyces pombe. A thorough understanding of fidelity mechanisms of DNA polymerases requires an analysis of the effects of sequence context and replication assessory proteins on fidelity. The proposed experiments, which include a full complement of polymerase subunits, are among the first of its kind, and make use of a mathematical model of polymerase fidelity and a gel fidelity assay that we've developed previously. The model is used to predict the effect of polymerase processivity subunits on base substitution fidelity. These predictions will be tested experimentally. Steady state kinetic experiments are designed to investigate the biochemical basis of mutational "hot" and "cold" spots. We propose to test the importance of hydrogen bonds between Watson-Crick base pairs on polymerase fidelity, by measuring the effects of base stacking on polymerase fidelity, in the absence of hydrogen bonding. Presteady state kinetic experiments, using fluorescent nucleotide analogs, are proposed to measure switching between polymerase and exonuclease active sites in "real-time". A second set of presteady state experiments are designed to determine the mechanism for loading and unloading the polymerase processivity clamp subunit onto DNA and to analyze the requirements for ATP hydrolysis for each step in the clamp-loading pathway. This pathway is required for Okazaki fragment formation during discontinuous lagging-strand DNA synthesis.

该资助提案的总体目标是研究 DNA 合成保真度的机制。 DNA聚合酶是参与DNA复制和修复的关键酶。 分析聚合酶如何控制保真度对于理解多种遗传疾病的生化基础至关重要。 Lesch-Nyhan 综合征和 ADA 缺乏症是由单点突变引起的遗传性儿童疾病的两个例子。 DNA 中单碱基的变化可能导致致癌基因的激活和抑癌基因的失活，从而导致癌症。 DNA聚合酶错误的复制后错配修复中的遗传缺陷是遗传性非息肉病科林癌以及多种其他类型癌症的根本原因。 先前的保真度研究主要集中在缺乏维持持续合成所需的聚合酶辅助蛋白的情况下的单个 DNA 聚合酶。 该资助研究了纯化的原核和真核 DNA 聚合酶全酶、大肠杆菌的 pol III 和 pol II 以及粟酒裂殖酵母的 pol δ 的保真度。 要全面了解 DNA 聚合酶的保真度机制，需要分析序列背景和复制评估蛋白对保真度的影响。 所提出的实验包括完整的聚合酶亚基，是此类实验中的第一个，并利用了我们之前开发的聚合酶保真度数学模型和凝胶保真度测定。 该模型用于预测聚合酶持续合成亚基对碱基取代保真度的影响。 这些预测将得到实验检验。 稳态动力学实验旨在研究突变“热点”和“冷”点的生化基础。 我们建议通过在没有氢键的情况下测量碱基堆积对聚合酶保真度的影响来测试 Watson-Crick 碱基对之间的氢键对聚合酶保真度的重要性。 建议使用荧光核苷酸类似物进行前稳态动力学实验，以“实时”测量聚合酶和核酸外切酶活性位点之间的切换。 第二组前稳态实验旨在确定将聚合酶持续钳亚基加载和卸载到 DNA 上的机制，并分析钳加载路径中每个步骤对 ATP 水解的要求。 在不连续的滞后链 DNA 合成过程中，冈崎片段的形成需要该途径。