Mechanistic Analysis of Pol Beta and Cancer-Associated Mutants

Pol Beta 和癌症相关突变体的机制分析

基本信息

批准号：
7464339
负责人：
MYRON GOODMAN
金额：
$ 42.91万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2013-07-13
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7464339
关键词：
2-Aminopurine Active Sites Address Affect Amino Acid Substitution Amino Acids Antineoplastic Agents Base Excision Repairs Base Pairing Biological Assay Biological Models Bone Tissue Cancer Etiology Cell model Charge Complex Computer Analysis Computer Simulation Coupled Cultured Cells DNA DNA Polymerase beta DNA biosynthesis DNA chemical synthesis DNA-Directed DNA Polymerase Data Dinucleotide Repeats Drug Design Electrostatics Excision Exhibits Fluorescence Frameshift Mutation Free Energy Genes Genetic Genomic Instability Germanium Goals Human In Vitro Individual Intervention Isotopes Kinetics LacZ Genes Left Link Lyase Malignant - descriptor Malignant Neoplasms Measures Modeling Motion Mutagenesis Mutation Nucleotides Oxygen Pharmacologic Substance Phenotype Phosphorus Plant Roots Play Polyacrylamide Gel Electrophoresis Polymerase Prevention strategy Production Property Proteins Reaction Role Series Simulate Site Source Structure System Techniques Testing Theoretical model Time Tissues Universities Variant analog base carbene carcinogenesis computer studies design in vitro Model inhibitor/antagonist interest metaplastic cell transformation millisecond mouse model mutant novel pol genes polymerization programs repaired research study size stopped-flow fluorescence tumor tumor progression

项目摘要

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.

项目 3 的总体目标是研究负责 DNA 合成和保真度的机制。从而解决有关突变（癌症的根本原因）的最基本问题。我们的具体方法是研究DNA聚合酶β（一种关键的修复聚合酶）的保真度。的变体 pol beta 与基因组不稳定和人类癌症有关。项目 3 的独特之处在于将其具体目标与 Pol beta 结构表征的建议紧密结合起来项目 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掺入正确和错误脱氧核苷酸之间的选择基材。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着关键作用。项目 3 研究 dNTP 底物过渡态类似物提供有关可用自由能源来源的新机械信息，以实现聚合酶来区分正确与错误。主要实验方法涉及使用荧光和快速淬灭前稳态动力学技术来测量整体保真度以及单独的保真度碱基替换和移码保真度组件。项目3将研究遗传通过构建体外系统模型来研究链位移的影响，更广泛地研究不稳定性合成单核苷酸和二核苷酸重复序列的扩展，产生移码突变，导致癌症。一般来说，项目项目，更具体地说，是实验 3，是及时给出的人们对 DNA 聚合酶在致癌中的作用重新产生了兴趣。实验3的研究过渡态类似物，与结构和计算项目结合起来，应该提供抗癌药物设计的实际回报，并提供设计的逻辑框架抑制癌症进展的药物干预和预防策略。项目 3 具有新功能财团合作者，耶鲁大学的 Joann Sweasy，他将鉴定和表征与人类肿瘤相关的实验 5 中的 pol beta 变体。