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的广泛目的是研究负责DNA合成忠诚和因此，要解决有关诱变的最基本问题，这是癌症的根本原因。我们的具体方法是研究DNA聚合酶β（一种钥匙修复聚合酶）的保真度。变体 POL Beta与基因组不稳定性和人类癌有关。项目3的独特方面是将其特定目的与为POL Beta的结构表征的特定目标紧密整合项目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合并正确与错误的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中起关键作用。项目3调查DNTP底物过渡状态类似于提供有关自由能源的新机械信息，可用于启用聚合酶以区分权利而不是错误。主要的实验方法涉及使用荧光和快速淬灭先进状态动力学技术，以测量整体保真度和个人保真度基础替代和移码保真度成分。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关于单核苷酸重复序列的扩展的合成，产生了移码突变引起癌症。程序项目一般，更具体地实验3，及时及时对DNA聚合酶在引起癌症中的作用的兴趣复兴。实验3中的研究与结构和计算项目一起进行的过渡状态类似物应提供药品抗癌药物设计的实用收益，并提供一个逻辑框架设计药物干预和预防策略抑制癌症的进展。项目3具有新的财团合作者，耶鲁大学乔安·斯威西（Joann Sweasy 实验5中的POL BETA变体。