Mechanism of Eukaryotic Environmental Mutagenesis

真核环境诱变机制

• 批准号: 8293540
• 负责人: GRAHAM C WALKER
• 金额: $ 43.14万
• 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/8293540
• 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; Screening procedure; 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; 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. PUBLIC HEALTH RELEVANCE: The proposed research will offer insights into the key fundamental processes that enable cells to repair and tolerate damage to their genetic material in response to environmental mutagens. These processes are responsible for the mutations that lead to cancer and a variety of human genetic diseases. The proposed research should also lead to the development of new classes of drugs to inhibit these processes that could have significant therapeutic applications.

描述（由申请人提供）：本基金资助的研究对进行翻译合成（TLS）的特殊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，但其他的，特别是Rev1和Pol ？（Rev3/Rev7）在真核生物中，参与TLS的一个致突变分支，该分支负责暴露于辐射和DNA损伤化学剂导致的大多数突变。除了揭示TLS中某些蛋白-蛋白相互作用的关键重要性外，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，并提供了令人震惊的证据，表明可以抑制Rev1/Pol ？依赖的TLS可能对人类健康有非常有益的应用。该实验将对TLS的分子机制产生新的见解，并旨在确定一类通过抑制TLS起作用的新型药物。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聚合酶在体内化疗肿瘤中的作用。我们建议在酵母和哺乳动物细胞中采用平行方法进行实验，这将使我们对Rev1/Pol ？-依赖的TLS，同时允许我们开发高通量荧光偏振分析，用于筛选破坏Rev1-Rev7-Rev3相互作用的小分子。我们还计划测试Rev1/3/7的功能是否可以被螺旋钉接肽和基于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有力地支持了TLS聚合酶在线粒体DNA损伤耐受中发挥额外关键作用的可能性，这是一个重要的话题，因为许多人类疾病都与线粒体DNA突变相关。我们将确定哪些TLS聚合酶对线粒体DNA突变是重要的，以响应选定的DNA损伤剂，并检查Rev1， Pol ？阿宝呢？利用生物化学和遗传的方法来研究线粒体。我们在过去的进展阶段的结果为TLS在化疗肿瘤中的重要性提供了新的见解，我们将继续在这一重要的生物学背景下研究TLS，我们将继续使用小鼠模型来研究Rev1和Pol ？在线粒体和核突变中。我们将确定DNA损伤化疗期间小鼠癌症模型中发生的依赖于Rev1/3/7的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依赖于Rev1/3/7的TLS来承受DNA损伤和响应这种损伤而发生突变的能力。