Structural determinants of Pol theta function

Pol theta 函数的结构决定因素

• 批准号: 10468631
• 负责人: Sylvie Doublie
• 金额: $ 35.1万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10468631
• 关键词: ATP phosphohydrolase; Architecture; Biochemical; Biological; Biological Assay; Bypass; Caenorhabditis elegans; Cell Line; Cells; Chimeric Proteins; Complex; Crystallization; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair Enzymes; DNA analysis; DNA-Directed DNA Polymerase; Dimerization; Electron Microscopy; Elements; Enzymes; Evaluation; Exonuclease; Feedback; Future; Genomic Instability; Genomics; In Vitro; Kinetics; Knowledge; Laboratories; Length; Link; Malignant Neoplasms; Mammalian Cell; Mediating; Methods; Molecular; Monitor; Mutation Analysis; Nucleotides; Pathway interactions; Play; Polymerase; Process; Property; Protein Engineering; Proteins; Research; Roentgen Rays; Role; Site; Specificity; Structure; Substrate Interaction; Testing; Therapeutic; Variant; Work; base; biophysical techniques; cancer therapy; carcinogenesis; design; experimental study; helicase; insight; molecular imaging; multidisciplinary; mutant; nuclease; paralogous gene; programs; protein purification; reconstitution; repaired; single molecule; targeted cancer therapy

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.

项目总结 这个项目将研究哺乳动物dna聚合酶θ，这是dna修复的决定酶。 聚合酶Theta介导的末端连接(TMEJ)导致双链断裂。尽管在生物学上具有重要的意义 TMEJ及其与癌症的相关性，我们对其分子机制知之甚少，令人惊讶。POLθ是一家大型 蛋白质(哺乳动物细胞中为290 kDa)，具有与DNA相连的解旋酶样域的独特排列 聚合酶结构域。除聚合酶结构域(PORD)外，POLθ还具有一个解旋酶样结构域 (保持)和连接中心域(CenD)。 结构信息对于分析DNA聚合酶机制是必不可少的，特别是对于大型 多结构域酶，如POLθ。我们确定了聚合酶结构域(Pold)的第一个晶体结构。 POLθ，连同DNA和一个输入的核苷酸。结构显示了独特的特征，这有助于 解释聚合酶的一些性质。我们在聚合酶中定位了5个插入环 核酸外切酶结构域。我们提出了POD二聚化的证据，这可能在连接过程中起作用 两个DNA分子。 该项目旨在填补知识中的几个主要空白，需要帮助我们了解独特的 POLθ的活性和结构：(1)“插入”环在哺乳动物的POLθ中起什么作用(2) 解旋酶样域(Hold)功能的结构基础？(3)末端修剪是如何发生的 微同源选择？(4)POLθ分子如何利用特定的界面协调修复？在……里面 除了获得对TMEJ机制的基本了解外，这项研究还将揭示新的 靶向在癌症治疗中抑制POLθ的机会。 这些结构研究将在计划项目内与其他三个项目高度协调 具有补充实验方法的项目--分子的单分子表征 功能，使用全长蛋白质的活性分析，以及修复的细胞研究。底物、蛋白质和 实验将通过项目1、2和4进行设计，并通过Core A Protein进行反馈监控。 提纯将由核心B支持，细胞系构建将由核心C支持。我们的联合工作将提供 对TMEJ途径的无与伦比的洞察力，以及其机制如何影响其实现其生物学能力 角色。这些见解将对我们理解这一途径对基因组不稳定的贡献至关重要 以及对该途径作为癌症治疗的安全和有效靶点的评价。