Mechanism of Eukaryotic Environmental Mutagenesis

描述(由申请人提供)：由该基金资助的研究对执行跨损伤合成(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聚合酶能够非常准确地复制到特定的“同源”病变上，但其他一些聚合酶，特别是真核生物中的Rev1和PolDNA(Rev3/Rev7)，参与了Tls的一个突变分支，该分支导致了暴露在辐射和ζ破坏性化学物质中的大多数突变。除了揭示某些蛋白质-蛋白质相互作用在TLS中的重要作用外，我们的研究还表明TLS聚合酶可能在线粒体中发挥重要作用，并提供了显著的证据表明，能够抑制依赖于REV1/POLζ的TLS的作用的药物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，并旨在识别一类通过抑制TLS发挥作用的新药物。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聚合酶在体内正在接受化疗的肿瘤中的作用。建议在酵母和哺乳动物细胞中采用平行方法进行实验，这将使我们能够获得依赖Rev1/PolTLS的分子细节的基础知识，同时允许我们开发高通量荧光偏振分析来筛选破坏Rev1-Rev7-Rev3相互作用的小分子。我们还计划测试Rev1/3/7功能是否可以被α-螺旋装订的多肽和基于尖端siRNAs的方法破坏。目前文献中只有三篇文章描述了TLS聚合酶在线粒体中的作用，但我们在过去一段时间的研究结果有力地支持了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，这是一个重要的课题，因为与线粒体DNA突变相关的许多人类疾病。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，Pol1，ζ和 使用生化和遗传方法将POLη连接到线粒体。在过去的进展阶段，我们的结果对Tls在接受化疗的肿瘤中的重要性有了新的见解，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Pol1ζ在线粒体和核突变中的体内作用。我们将确定依赖Rev1/3/7的线粒体和核突变的性质，这些突变发生在DNA损毁化疗期间的小鼠癌症模型中，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Rev1/3/7的TLS来抵御DNA损伤和针对此类损伤进行突变的能力。

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 ζ (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 ζ-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 ζ-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 α-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 ζ, and Pol η 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 ζ 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.