Mechanism of Eukaryotic Environmental Mutagenesis

项目总结/摘要 一种基本的分子机制，几乎所有生物体都通过这种机制对环境损害作出反应， 它们的遗传物质是通过在DNA损伤上进行跨损伤合成（TLS）而获得的。真核 Rev 1/3/7依赖的诱变TLS途径对人类健康至关重要，这不仅是因为它可以 通过耐受环境DNA损伤，包括修复DNA交联，帮助细胞存活， 因为这一分子过程是导致绝大多数突变的原因 对DNA的损伤。来自环境暴露的突变会导致癌症，其他人类疾病， 衰老Rev 1是TLS DNA聚合酶的Y家族的成员，而Rev 3和Rev 7分别是TLS DNA聚合酶的Y家族的成员。 TLS DNA聚合酶的催化和非催化亚基。这项研究的总体目标是建立在令人兴奋的 通过利用人类和生物医学领域的新发展， 哺乳动物生物学获得详细的新见解的机制，调节，和生理 这一依赖于Rev 1/3/7的进程在决议水平上的后果， 使用具有复杂遗传系统的生物体，如大肠杆菌和酵母菌， 啤酒。这项研究的一个特别创新的组成部分是开发一套新的抑制剂， 干扰Rev 1/3/7依赖性TLS、DNA交联修复和其他Rev 1/3/7相关的 流程.这些不仅是推进生物体如何对环境做出反应的基础研究的有力探针， DNA从环境化学物质，但也有潜力改善化疗和可能的其他 人类健康方面。一个主要的策略是识别抑制Rev 1/3/7依赖性的小分子。 通过干扰途径运行所需的关键相互作用，如 Rev 1 100个氨基酸C-末端结构域（CTD）与DNA聚合酶Rev 7组分的相互作用 通过一个界面和其他TLS DNA聚合酶的RIR（Rev 1相互作用区）通过第二个界面 接口.已经鉴定了结合每个Rev 1界面并具有 预期的生物效应。这些将在人类肺癌的syngeic小鼠模型中进行评估， 淋巴瘤作为可能的化疗佐剂，增加杀伤，同时也减少诱变， 引起了抵抗。抑制Rev 1/3/7依赖性诱变TLS的其他创新方法包括 使用钉合的RIR肽，使用炭疽保护性抗原将Rev 1 CTD递送到哺乳动物中， 通过将其融合到致死因子的N末端，并测试Rev 7相互作用序列是否可以 作为显性负抑制剂通过捕获Rev 7在非生产性复合物。在一种补充方法中， 一系列影响Rev 1/3/7依赖性途径中蛋白质的部分功能丧失突变体将产生 详细的功能见解复杂的蛋白质编排，基础的关键作用，诱变 TLS在DNA损伤耐受性、交联修复和环境化学品诱变中的应用。

Project Summary/Abstract A fundamental molecular mechanism by which virtually all organisms respond to environmental damage to their genetic material is by carrying out translesion synthesis (TLS) over DNA lesions. The eukaryotic Rev1/3/7-dependent pathway of mutagenic TLS is critically important to human health, not only because it can help cells to survive by tolerating environmental DNA damage, including the repair of DNA crosslinks, but also because this molecular process is responsible for the vast majority of the mutagenesis that occurs as a result of damage to DNA. Mutations from environmental exposure contribute to cancer, other human diseases, and aging. Rev1 is member of the Y family of TLS DNA polymerases, while Rev3 and Rev7 respectively are the catalytic and non-catalytic subunits of TLS DNA pol ζ. The overall goal of this research is to build on exciting progress funded by NIEHS grant R01-ES015818 by taking advantage of new developments in human and mammalian biology to gain detailed new insights into the mechanism, regulation, and physiological consequences of this Rev1/3/7-dependent process at a level of resolution that has historically only been attainable using organisms with sophisticated genetic systems such as Escherichia coli and Saccharomyces cerevisiae. A particularly innovative component of this research is to develop a suite of novel inhibitors and other strategies to interfere with Rev1/3/7-dependent TLS, DNA crosslink repair, and other Rev1/3/7-related processes. These will not only be powerful probes to advance basic research into how organisms respond to DNA from environmental chemicals, but also have the potential to improve chemotherapy and possibly other aspects of human health. One major strategy is to identify small molecules that inhibit Rev1/3/7-dependent mutagenic TLS by interfering with critical interactions required for operation of the pathway, such as the interaction of the Rev1 100 amino acid C-terminal domain (CTD) with the Rev7 component of DNA pol ζ through one interface and the RIR (Rev1-interacting region) of other TLS DNA polymerases through a second interface. Exemplar compounds have already been identified that bind to each Rev1 Interface and have the expected biological effects. These will be evaluated in syngeic mouse models of human lung cancer and lymphoma as possible chemotherapy adjuvants that increase killing while also reducing the mutagenesis that gives rise to resistance. Other innovative approaches to inhibiting Rev1/3/7-dependent mutagenic TLS include using stapled RIR peptides, using the Anthrax Protective Antigen to deliver the Rev1 CTD into mammalian cells by fusing it to the N-terminus of Lethal Factor, and testing whether Rev7-interacting sequences can serve as dominant negative inhibitors by trapping Rev7 in nonproductive complexes. In a complementary approach, a series of partial-loss-of-function mutants affecting proteins in the Rev1/3/7-dependent-pathway will yield detailed functional insights into the complex protein choreography that underlies the crucial roles of mutagenic TLS in DNA damage tolerance, crosslink repair, and mutagenesis from environmental chemicals.