Mechanism of Eukaryotic Environmental Mutagenesis

真核环境诱变机制

• 批准号: 8446396
• 负责人: GRAHAM C WALKER
• 金额: $ 40.92万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446396
• 关键词: Affect; Biochemical; Biochemical Genetics; Biological; Biological Assay; Cells; Chemical Agents; DNA Damage; DNA Repair; DNA-Directed DNA Polymerase; Development; Employee Strikes; Eukaryota; Exposure to; Fluorescence Polarization; Funding; Genetic; Genetic Materials; Grant; Health; Hereditary Disease; Human; Human Genetics; Knowledge; Lead; Lesion; Literature; Malignant Neoplasms; Mammalian Cell; Mammals; Mitochondria; Mitochondrial DNA; Molecular; Mutagenesis; Mutate; Mutation; Nature; Nuclear; Paper; Peptides; Pharmaceutical Preparations; Play; Polymerase; Preclinical Drug Evaluation; Process; Proteins; Radiation; Research; Role; Small Interfering RNA; System; Testing; Therapeutic; Time; Work; Yeasts; base; chemotherapy; design; environmental mutagens; high throughput screening; human disease; improved; in vivo; insight; mouse model; protein protein interaction; repaired; research study; response; screening; small molecule; tumor; tumor progression

DESCRIPTION (provided by applicant): Research funded by this grant has yielded important new insights into the mechanism and biological roles of the specialized DNA polymerases that carry out translesion synthesis (TLS). Certain TLS polymerases are able to copy quite accurately over particular "cognate" lesions but others, notably Rev1 and Pol ? (Rev3/Rev7) in eukaryotes, participate in a mutagenic branch of TLS that is responsible for most of the mutations that result from exposure to radiation and DNA damaging chemical agents. In addition to revealing the critical importance of certain protein-protein interactions in TLS, our studies suggest that TLS polymerases may also play important roles in the mitochondria and provide striking evidence suggesting that drugs that could suppress the action of Rev1/Pol ? -dependent TLS could have very beneficial applications for human health. The proposed experiments will yield new insights into the molecular mechanism of TLS and are designed to identify a new class of drug that acts by inhibiting TLS. They will also evaluate the roles of TLS polymerases in mitochondria and further characterize the roles of TLS polymerases in tumors that are undergoing chemotherapy in vivo. Experiments are proposed involving parallel approaches in both yeast and mammalian cells that should allow us to gain fundamental knowledge into the molecular details of Rev1/Pol ?-dependent TLS, while at the same time allowing us to develop high-throughput fluorescence polarization assays for screening for small molecules that disrupt Rev1-Rev7-Rev3 interactions. We also plan to test whether Rev1/3/7 function can be disrupted by ¿-helical stapled peptides and by cutting-edge siRNA-based approaches. There are presently only three papers in the literature describing the roles of TLS polymerases in the mitochondria, however our results during the past progress period strongly support the possibility that TLS polymerases play additional key role in DNA damage tolerance in the mitochondria, an important topic because of the many human diseases associated with mutations in mitochondrial DNA. We will determine which TLS polymerases are important for mitochondrial DNA mutagenesis in response to selected DNA damaging agents and examine the localization of Rev1, Pol ?, and Pol ? to the mitochondria using both biochemical and genetic approaches. Our results during the past progress period have yielded new insights into the importance of TLS in tumors undergoing chemotherapy, and we will continue to study TLS in this important biological context and we will continue to use mouse models to investigate the in vivo roles of Rev1 and Pol ? in mitochondrial and nuclear mutagenesis. We will determine the nature of the Rev1/3/7-dependent mitochondrial and nuclear mutations occurring in mouse models of cancer during DNA damaging chemotherapy and investigate how key mutations associated with cancer progression and DNA repair/checkpoints affect a cell's ability to use Rev1/3/7 dependent TLS to withstand DNA damage and to mutate in response to such damage.

描述（由申请人提供）：由该基金资助的研究对进行跨病变合成（TLS）的专门DNA聚合酶的机制和生物学作用产生了重要的新见解。某些TLS聚合酶能够相当准确地复制特定的“同源”病变，但其他人，特别是Rev 1和Pol？(Rev3/Rev 7）在真核生物中，参与TLS的致突变分支，其负责由暴露于辐射和DNA损伤化学试剂引起的大多数突变。除了揭示TLS中某些蛋白质-蛋白质相互作用的至关重要性外，我们的研究表明TLS聚合酶也可能在线粒体中发挥重要作用，并提供了惊人的证据表明，可以抑制Rev 1/Pol？依赖TLS可能对人类健康有非常有益的应用。拟议的实验将对TLS的分子机制产生新的见解，并旨在确定一类通过抑制TLS发挥作用的新药。他们还将评估TLS聚合酶在线粒体中的作用，并进一步表征TLS聚合酶在体内接受化疗的肿瘤中的作用。实验提出涉及酵母和哺乳动物细胞的平行方法，应该让我们获得基本知识的分子细节Rev 1/Pol？依赖性TLS，同时允许我们开发高通量荧光偏振分析，用于筛选破坏Rev 1-Rev 7-Rev 3相互作用的小分子。我们还计划测试Rev 1/3/7功能是否可以被半螺旋钉合肽和基于siRNA的尖端方法破坏。目前文献中只有三篇论文描述了TLS聚合酶在线粒体中的作用，然而我们在过去的进展期间的结果强烈支持TLS聚合酶在线粒体中DNA损伤耐受性中发挥额外关键作用的可能性，这是一个重要的主题，因为许多人类疾病与线粒体DNA突变相关。我们将确定哪种TLS聚合酶对线粒体DNA诱变响应选定的DNA损伤剂是重要的，并检查Rev 1，Pol？，还有波尔应用生物化学和遗传学的方法来研究线粒体。我们的研究结果在过去的进展期间已经产生了新的见解TLS在肿瘤化疗的重要性，我们将继续研究TLS在这一重要的生物背景下，我们将继续使用小鼠模型来调查在体内的作用Rev 1和Pol？在线粒体和核诱变中。我们将确定在DNA损伤化疗期间发生在小鼠癌症模型中的Rev 1/3/7依赖性线粒体和核突变的性质，并研究与癌症进展和DNA修复/检查点相关的关键突变如何影响细胞使用Rev 1/3/7依赖性TLS的能力来承受DNA损伤并响应于这种损伤而突变。