Structural determinants of Pol theta function

项目摘要 本计画将研究哺乳动物DNA聚合酶θ，它是DNA修复的定义酶 通过聚合酶θ介导的末端连接（TMEJ）的双链断裂。尽管生物学上的重要性， TMEJ及其与癌症的相关性，我们对其分子机制知之甚少。Pol θ是一个大的 一种蛋白质（在哺乳动物细胞中为290 kDa），具有与DNA连接的解旋酶样结构域的独特排列 聚合酶结构域。除了聚合酶结构域（PolD）外，Pol θ还具有解旋酶样结构域 （HelD）和连接中心结构域（CenD）。 结构信息对于分析DNA聚合酶机制是必不可少的，特别是对于大的 多结构域酶如Pol θ。我们确定了聚合酶结构域（PolD）的第一晶体结构， 与DNA和一个新的核苷酸一起。结构显示出独特的特征，这有助于 解释聚合酶的一些特性。我们在聚合酶中定位了5个插入环， 外切核酸酶结构域。我们提出了PolD二聚化的证据，它可以在连接过程中发挥作用。 两个DNA分子。 这个项目旨在填补知识的几个主要空白，需要帮助我们了解独特的 Pol θ的活性和结构：（1）哺乳动物Pol θ的“插入”环起什么作用？（2）哺乳动物Pol θ的“插入”环是什么？ 解旋酶样结构域（HelD）功能的结构基础？(3)如何进行末端修剪， 微同源选择(4)Pol θ分子如何利用特定的界面协调修复？在 除了获得对TMEJ机制的基本了解外，这项研究还将揭示新的 在癌症治疗中抑制Pol θ的靶向机会。 这些结构研究将在计划项目内与其他三个项目高度协调。 具有互补实验方法的项目-分子的单分子表征 功能，使用全长蛋白质的活性测定，以及修复的细胞研究。底物、蛋白质和 实验将与项目1、2和4一起设计，并通过核心A进行反馈监测。蛋白 核心B支持纯化，核心C支持细胞系构建。我们的联合工作将提供 对TMEJ途径的无与伦比的洞察，以及其机制如何影响其实现生物学功能的能力， 作用这些见解将是至关重要的，我们了解的贡献，这一途径的基因组不稳定性 和致癌作用，以及评估该途径作为癌症治疗的安全有效靶点。

PROJECT SUMMARY This project will investigate mammalian DNA polymerase θ, the defining enzyme for repair of DNA double-strand breaks by polymerase theta-mediated end joining (TMEJ). Despite the biological importance of TMEJ and its relevance to cancer, we know surprisingly little about its molecular mechanisms. Pol θ is a large protein (290 kDa in mammalian cells) with a distinctive arrangement of a helicase-like domain linked to a DNA polymerase domain. In addition to the polymerase domain (PolD), Pol θ possesses a helicase-like domain (HelD) and a connecting central domain (CenD). Structural information is essential for analyzing DNA polymerase mechanisms, especially for a large multi-domain enzyme such as Pol θ. We determined the first crystal structure of the polymerase domain (PolD) of Pol θ, together with DNA and an incoming nucleotide. The structure revealed unique features, which help explain some of the properties of the polymerase. We located 5 insertion loops in the polymerase and pseudo- exonuclease domains. We presented evidence for dimerization of the PolD, which could function during joining of two DNA molecules. This project aims to fill several major gaps in knowledge, needed to help us understand the unique activities and structure of Pol θ: (1) What role do the “insertion” loops play in mammalian Pol θ (2) What is the structural basis of helicase-like domain (HelD) function? (3) How does end-trimming occur during microhomology selection? (4) How do molecules of Pol θ coordinate repair, using specific interfaces? In addition to gaining a fundamental understanding of the TMEJ mechanism, the research will reveal new targeting opportunities for Pol θ inhibition in cancer therapy. These structural studies will be highly coordinated within the Program Project with the other three Projects with complementary experimental approaches – single-molecule characterization of molecular function, activity assays using full-length proteins, and cellular studies of repair. Substrates, proteins, and experiments will be designed with Projects 1, 2, and 4 and monitored with feedback via Core A. Protein purification will be supported by Core B, and cell line construction by Core C. Our combined work will provide unparalleled insight into the TMEJ pathway, and how its mechanism impacts its ability to fulfill its biological role. These insights will be critical to our understanding of the contribution of this pathway to genome instability and carcinogenesis, as well as the evaluation of this pathway as a safe and effective target for cancer therapy.