Structural determinants of Pol theta function

项目概要 该项目将研究哺乳动物 DNA 聚合酶 θ，这是 DNA 修复的定义酶 聚合酶 theta 介导的末端连接 (TMEJ) 导致双链断裂。尽管具有生物学重要性 TMEJ 及其与癌症的相关性，令人惊讶的是我们对其分子机制知之甚少。 Pol θ 是一个很大的 蛋白质（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) 之外，Pol θ 还具有解旋酶样结构域 (HelD) 和连接中心域 (CenD)。 结构信息对于分析 DNA 聚合酶机制至关重要，尤其是对于大型 DNA 聚合酶来说。 多域酶，例如 Pol θ。我们确定了聚合酶结构域 (PolD) 的第一个晶体结构 Pol θ，以及 DNA 和传入的核苷酸。该结构揭示了独特的功能，这有助于 解释聚合酶的一些特性。我们在聚合酶和伪-中定位了5个插入环 核酸外切酶结构域。我们提供了 PolD 二聚化的证据，该二聚化可以在连接过程中发挥作用 两个 DNA 分子。 该项目旨在填补几个主要的知识空白，帮助我们了解独特的知识 Pol θ 的活动和结构： (1) “插入”环在哺乳动物 Pol θ 中起什么作用 (2) 什么是 Pol θ 解旋酶样结构域（HelD）功能的结构基础？ (3) 切边是如何发生的 微同源选择？ (4) Pol θ 分子如何利用特定的界面进行协调修复？在 除了获得对 TMEJ 机制的基本了解外，该研究还将揭示新的 寻找癌症治疗中 Pol θ 抑制的机会。 这些结构研究将在该计划项目内与其他三个研究高度协调 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 实验将根据项目 1、2 和 4 进行设计，并通过 Core A 进行反馈监测。 蛋白质 纯化将由 Core B 支持，细胞系构建将由 Core C 支持。我们的联合工作将提供 对 TMEJ 通路及其机制如何影响其实现其生物学功能的能力具有无与伦比的洞察力 角色。这些见解对于我们理解该途径对基因组不稳定的贡献至关重要 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.