Mechanistic Analysis of Pol Beta and Cancer-Associated Mutants

项目3的主要目标是研究负责DNA合成保真度的机制， 从而解决有关突变的最基本问题，这是癌症的根本原因。我们 具体的方法是研究DNA聚合酶β（一种关键的修复聚合酶）的保真度。各种 pol β与基因组不稳定性和人类癌症有关。项目3的独特之处在于， 将其具体目标与为Pol β的结构表征而提出的目标紧密结合， 项目1和项目2中的理论计算研究，我们可以测试定量预测 活性位点氨基酸如何控制正确和错误脱氧核苷酸的选择 印刷受体.通过对理论计算和结构预测进行严格的测试，数据将 在完善理论模型中发挥关键作用。项目3研究dNTP底物过渡态 类似物，以提供关于自由能来源的新的机械信息， 聚合酶来区分对错。主要的实验方法包括使用 荧光和快速淬灭预稳态动力学技术来测量整体保真度以及 单个保真度碱基取代和移码保真度组分。项目3将研究基因 通过构建体外系统模型来研究链置换的影响， 扩增单和二核苷酸重复序列的合成，产生移码突变， 导致癌症。一般来说，计划项目，更具体地说，实验3及时给出了 对DNA聚合酶在致癌中的作用的兴趣重新抬头。实验3中， 过渡态类似物，结合结构和计算项目，应提供 在制药抗癌药物设计的实际回报，并提供了一个逻辑框架，设计 药物干预和预防策略，以抑制癌症进展。项目3具有新的 联盟合作者，Joann Sweasy，耶鲁大学，他将识别和表征人类肿瘤相关的 实验5中的pol β变体。

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.