ERROR CORRECTION IN DNA SYNTHESIS--A BIOCHEMICAL STUDY

这项拨款提案的广泛目标是研究负责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是理解各种遗传疾病的生化基础的核心。Lesch-Nyhan综合征和ADA缺乏症是可由单点突变引起的遗传性儿童疾病的两个例子。致癌基因的激活和肿瘤抑制基因的失活导致癌症可以由DNA的单碱基改变引起。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性结肠癌的根本原因，以及各种其他类型的癌症。以前的保真度研究主要集中在缺乏维持过程合成所需的聚合酶辅助蛋白的单个DNA聚合酶上。本基金研究纯化的原核生物和真核生物DNA聚合酶全酶、大肠杆菌的pol III和pol II以及pombe Schizosaccharomyces的pol delta的保真度。彻底了解DNA聚合酶的保真机制需要分析序列背景和复制评估蛋白对保真度的影响。提议的实验，包括聚合酶亚基的完整补充，是同类实验中的第一个，并利用了聚合酶保真度的数学模型和我们之前开发的凝胶保真度测定。该模型用于预测聚合酶加工亚基对碱基取代保真度的影响。这些预测将在实验中得到验证。稳态动力学实验旨在研究突变“热点”和“冷点”的生化基础。我们建议在没有氢键的情况下，通过测量碱基堆叠对聚合酶保真度的影响，来测试沃森-克里克碱基对之间氢键对聚合酶保真度的重要性。采用荧光核苷酸类似物的前稳态动力学实验，提出了“实时”测量聚合酶和外切酶活性位点之间切换的方法。第二组预稳态实验旨在确定在DNA上加载和卸载聚合酶加工钳亚基的机制，并分析钳加载途径中每个步骤对ATP水解的要求。该途径是在不连续滞后链DNA合成过程中形成冈崎片段所必需的。

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.