Structural determinants of Pol theta function

Pol theta 函数的结构决定因素

基本信息

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
项目类别：
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

项目摘要

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，DNA修复的防御酶尽管生物学的重要性，但通过聚合酶的双链破裂（TMEJ）。 TMEJ及其与癌症的相关性，我们对其分子机制的了解很少。蛋白质（290 kDa在mamalian细胞中），具有解旋酶样结构域的独特排列到DNA 除了聚合酶结构域（POLD）之外，聚合酶结构域还具有解旋酶样域。（持有）和连接的中央域（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。 polθ，以及DNA和收入的核苷酸。解释一些亲A聚合酶。外核酸酶域。两个DNA分子。该项目旨在填补知识的几个主要空白，以帮助您实现独特 POLθ的活动和结构：（1）“插入”环在哺乳动物polθ（2）中扮演什么角色。解旋酶样域（HEW）功能的结构基础？微型学的选择？除了获得对TMEJ机制的基本理解外，该研究还将陶醉 Targeting Opportunities for Pol Pol θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θl θlθlθlθlθlθlθlθlθlθlθlθlθlθlθlθlθlθlθ 这些结构研究将在计划项目中与其他三棵树进行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，使用全长蛋白的活性测定以及修复的细胞研究。通过项目1、2和4和4进行设计，并通过核心核心A的反馈对反馈进行监控。蛋白质纯化将由Core B支持，并由CoreC进行细胞系构建。我们的合并工作提供了对TMEJ途径的无与伦比的洞察力，Howes机制会影响其生物学这些见解对我们对theis途径贡献的贡献的理解至关重要和癌变，以及评估作为癌症治疗的安全有效靶标的评估。