Mechanistic Analysis of Pol Beta and Cancer-Associated Mutants

项目3的主要目标是研究DNA合成和DNA合成保真度的机制。 从而解决与突变有关的最基本的问题，突变是癌症的根本原因。我们的 具体的方法是调查DNA聚合酶β的保真度，这是一种关键的修复聚合酶。的变种 POLβ与基因组不稳定和人类癌症有关。项目3的独特之处在于，通过 将其具体目标与建议的POLβ结构表征紧密结合在一起 项目1和项目2中的理论计算研究，我们可以检验定量预测 活性部位氨基酸如何支配对错脱氧核苷酸的选择 底物。通过对理论计算和结构预测进行严格的测试，数据将 在完善理论模型方面发挥了关键作用。项目3研究dNTP底物过渡态 类似物，以提供关于可用于实现的自由能量来源的新的机械信息 聚合酶用来区分对错。主要的实验方法涉及使用 测量整体保真度的荧光和快速猝灭稳态前动力学技术以及 单独的保真度基准替换和移帧保真度组件。项目3将调查基因 更一般地通过构建体外系统模型来研究链移位的影响 扩增产生移码突变的单核苷酸和二核苷酸重复序列的合成 致癌。计划项目一般，更具体地说，实验3，是及时给予 人们对DNA聚合酶在致癌中的作用重新产生了兴趣。实验3中关于 过渡态模拟，结合结构和计算项目，应该提供 在药物抗癌药物设计中的实际收益，并提供了设计的逻辑框架 抑制癌症进展的药物干预和预防策略。Project 3采用了一个新的 该联盟的合作者，耶鲁大学的Joann Sweasy，他将识别和描述与人类肿瘤相关的 实验5中的POL Beta变种。

The broad objective of Project 3 is to study the mechanisms responsible for the fidelity of DNA synthesis and thus to address the most fundamental questions concerning mutagenesis, a root cause of cancer. Our specific approach is to investigate the fidelity of DNA polymerase beta, a key repair polymerase. Varients of pol beta are associated with genome instability and human cancer. The unique aspect of Project 3 is that by closely integrating its specific aims with those proposed for the structural characterization of Pol beta in Project 1 and the the theoretical computational study in Project 2, we can test quantitative predictions for how active site amino acids govern the choice between incorporating right and wrong deoxynucleotide substrates. By providing a stringent test of theoretical-computational and structural predictions, the data will play a key role in refining the theoretical models. Project 3 investigates dNTP substrate transition state analogs to provide new mechanistic information concerning the source of free energy available to enable polymerases to distinguish right from wrong. The main experimental approach involves the use of fluorescence and rapid quench presteady state kinetic techniques to measure overall fidelity as well as individual fidelity base substitution and frameshift fidelity components. Project 3 will investigate genetic instability more generally by constructing model in vitro systems to study the effects of strand displacement synthesis on the expansion of mono- and dinucleotide repeat sequences yielding frameshift mutation that cause cancer. The Program Project generally, and Experiment 3 more specifically, are timely given the resurgence of interest in the role of DNA polymerases in causing cancer. The studies in Experiment 3 on transition state analogs, taken in conjunction with the structural and computational projects, should provide practical payoffs in pharmaceutical anticancer drug design, and offer a logical framework in which to design drug intervention and prevention strategies to inhibit cancer progression. Project 3 features a new consortium collaborator, Joann Sweasy, Yale University, who will indentify and characterize human tumorassociated pol beta variants in Experiment 5.