ERROR CORRECTION IN DNA SYNTHESIS--A BIOCHEMICAL STUDY

这项拨款提案的主要目标是研究负责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缺乏症是两个遗传性儿童疾病的例子，可以从一个单一的点突变。 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中的单个碱基变化引起。 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，沿着多种其他类型的癌症。 以前的保真度研究集中在个别的DNA聚合酶的聚合酶辅助蛋白的情况下，需要维持进行性合成。 这项资助研究了纯化的原核和真核DNA聚合酶全酶的保真度，大肠杆菌的聚合酶III和聚合酶II，以及裂殖酵母的聚合酶δ。 深入了解DNA聚合酶的保真度机制需要分析序列背景和复制辅助蛋白对保真度的影响。 所提出的实验，其中包括一个完整的聚合酶亚基，是第一个同类的，并利用聚合酶保真度的数学模型和凝胶保真度测定，我们以前已经开发。 该模型用于预测聚合酶持续合成能力亚基对碱基取代保真度的影响。 这些预测将在实验中得到检验。 稳态动力学实验旨在研究突变“热”和“冷”点的生化基础。 我们建议测试沃森-克里克碱基对之间的氢键对聚合酶保真度的重要性，通过测量的影响，在没有氢键的情况下，聚合酶保真度的碱基堆积。 提出了利用荧光核苷酸类似物的预稳态动力学实验来“实时”测量聚合酶和核酸外切酶活性位点之间的切换。 设计第二组预稳态实验以确定将聚合酶持续合成能力钳亚基装载和卸载到DNA上的机制，并分析钳装载途径中每个步骤对ATP水解的要求。 该途径是不连续滞后链DNA合成过程中冈崎片段形成所必需的。

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.