Structural determinants of Pol theta function

Pol theta 函数的结构决定因素

• 批准号: 10640895
• 负责人: Sylvie Doublie
• 金额: $ 34.45万
• 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/10640895
• 关键词: ATP phosphohydrolase; Architecture; Binding; Biochemical; Biological; Biological Assay; Bypass; Caenorhabditis elegans; Cell Line; Cells; Chimeric Proteins; Complex; 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; biophysical techniques; cancer therapy; carcinogenesis; design; experimental study; helicase; insight; molecular imaging; multidisciplinary; mutant; nuclease; paralogous gene; programs; protein purification; reconstitution; repaired; single molecule; structural determinants; 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.

项目摘要 该项目将研究哺乳动物DNA聚合酶θ，该酶是修复DNA的定义酶 通过聚合酶theta介导的末端连接（TMEJ）的双链断裂。尽管生物学的重要性 TMEJ及其与癌症的相关性，我们对其分子机制知之甚少。 polθ是一个大的 蛋白质（哺乳动物细胞中的290 kDa）具有与DNA相关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，polθ具有类似解旋酶的结构域 （持有）和一个连接的中央域（Cend）。 结构信息对于分析DNA聚合酶机制至关重要，特别是对于大型 多域酶，例如polθ。我们确定了聚合酶结构域（POLD）的第一个晶体结构 polθ，以及DNA和传入的核苷酸。该结构揭示了独特的功能，这有帮助 解释聚合酶的某些特性。我们在聚合酶和伪 - 中找到了5个插入环。 外切核酸酶域。我们提供了pold二聚体的证据，这些证据可以在加入过程中起作用 两个DNA分子。 该项目旨在填补知识的几个主要空白，以帮助我们了解独特的知识 POLθ的活动和结构：（1）“插入”环在哺乳动物polθ中扮演什么角色（2） 解旋酶样域（HEW）功能的结构基础？ （3）在 微学选择？ （4）使用特定界面如何使用POLθ坐标修复分子？在 除了获得对TMEJ机制的基本了解，该研究还将揭示新的 靶向癌症治疗中的polθ抑制机会。 这些结构研究将在计划项目中与其他三个研究进行高度协调 具有完成实验方法的项目 - 分子的单分子表征 功能，使用全长蛋白的活性评估以及修复的细胞研究。底物，蛋白质和 实验将使用项目1、2和4设计，并通过核心A蛋白质对反馈进行监控 纯化将由核心B支持，并由CoreC进行细胞系结构。我们的合并工作将提供 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的能力 角色。这些见解对于我们对这一途径对基因组不稳定性的贡献的理解至关重要 和癌变，以及对该途径的评估，作为癌症治疗的安全有效靶标。