Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity

布氏锥虫 TRF 维持端粒完整性的机制

• 批准号: 10622535
• 负责人: Bibo Li
• 金额: $ 18.65万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622535
• 关键词: Affect; Air; Antigenic Switching; Antigenic Variation; Atomic Force Microscopy; Automobile Driving; Binding; Biological Assay; Biology; Cells; Chromosomes; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA ligase I; Development; ERCC1 gene; Electrophoretic Mobility Shift Assay; Event; Exhibits; Frequencies; Future; Genes; Genetic; Genetic Recombination; Genetic Transcription; Homodimerization; Image; Immune response; Immunologics; In Vitro; Lead; Leishmania; Liquid substance; Mammals; Mediating; Membrane Glycoproteins; Molecular; Nonhomologous DNA End Joining; North Carolina; Nucleic Acids; Parasites; Parasitic infection; Pathogenesis; Pathway interactions; Play; Proteins; RNA; RNA Binding; RNA Interference; RNA Polymerase I; Rad51 recombinase; Role; Site; Southern Blotting; Speed; Structure; Surface Antigens; Testing; Trypanosoma brucei brucei; Trypanosoma cruzi; Universities; Variant; dimer; gel electrophoresis; homologous recombination; human disease; in vivo; knock-down; microscopic imaging; mutant; novel; novel therapeutics; pathogen; pathogenic microbe; rare variant; recombinase; recruit; single molecule; telomere; tool

Project Summary/Abstract Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are closely related kinetoplastid parasites causing debilitating human diseases. T. brucei sequentially expresses immunologically distinct variant surface glycoproteins (VSGs), its major surface antigen, exclusively from the subtelomeric VSG expression sites (ESs) to evade the host immune response. Similarly, a number of other eukaryotic pathogens that undergo antigenic variation also express their major surface antigens from subtelomeres, and DNA recombination is an important means of antigen switching. Studies of T. brucei telomere biology have shown that perturbation of the telomere structure can be a double-edged sword: increasing telomere stability suppresses VSG switching, while losing gene integrity at the active VSG vicinity results in nearly 90% of cell lethality. We have shown that TRF, the duplex telomere DNA binding factor, plays important roles in maintaining telomere integrity and stability. The active VSG-adjacent telomere is transcribed by RNA Polymerase I into TERRA, which can form the telomeric R-loop (TRL) with the telomeric DNA. TRF suppresses TERRA and TRL levels, and more TRLs in TRF- depleted cells lead to more telomeric DNA damage. However, the underlying mechanisms are unclear. Homologous Recombination (HR) is a major VSG switching pathway, yet deletion of the key HR recombinase RAD51 does not eliminate recombination-mediated switching, indicating that other recombination mechanisms are involved. Microhomology-Mediated End Joining (MMEJ) events exist in T. brucei, but it is unknown whether VSG switching can occur through MMEJ. Antigenic variation is an essential pathogenesis mechanism enabling a long-term parasite infection. Understanding how telomere proteins affect VSG switching and identification of all switching pathways will help us develop means to eradicate this parasite in the future. To better understand how telomere proteins affect VSG switching and to identify additional recombination mechanisms involved in antigenic variation, we will investigate how TRF helps maintain telomere integrity and stability using novel single-molecule analyses – Atomic Force Microscopy imaging (AFM, in air and high-speed in liquids) and DNA tightropes – and genetic and molecular tools through the following aims. In Aim 1, we will examine how TRF suppresses TRL by testing whether TRF binds TRL directly and whether TRF can recruit TERRA to the duplex telomeric DNA by binding to both nucleic acids through different TRF molecules and homodimerization. We will also examine the TERRA localization and R-loop levels in TRF point mutants that weaken or enhance its TERRA binding activity. In Aim 2, we will take advantage that TRF-depleted cells have more recombination products and examine whether HR and MMEJ contribute to telomeric/ subtelomeric instability by deleting/knockdown factors essential for these pathways in TRF RNAi cells. We will then examine VSG switching in cells lacking key recombination players in the WT TRF background. Our studies will reveal how TRF maintains telomere integrity and identify potential additional important factors in antigenic variation.

项目概要/摘要 布氏锥虫、克氏锥虫和利什曼原虫是密切相关的动质体寄生虫 导致人类衰弱的疾病。 T. brucei 依次表达免疫学上不同的变异表面 糖蛋白 (VSG)，其主要表面抗原，完全来自亚端粒 VSG 表达位点 (ES) 以逃避宿主的免疫反应。类似地，许多其他真核病原体也经历抗原性 变异还表达来自亚端粒的主要表面抗原，DNA重组是重要的 抗原转换的手段。对布氏锥虫端粒生物学的研究表明，端粒的扰动 结构可以是一把双刃剑：增加端粒稳定性会抑制 VSG 转换，同时失去端粒稳定性 活性 VSG 附近的基因完整性导致近 90% 的细胞致死率。我们已经证明，TRF 双链体端粒DNA结合因子，在维持端粒完整性和稳定性方面发挥着重要作用。这 活性 VSG 相邻端粒由 RNA 聚合酶 I 转录成 TERRA，从而形成端粒 带有端粒 DNA 的 R 环 (TRL)。 TRF 抑制 TERRA 和 TRL 水平，以及 TRF 中更多的 TRL- 耗尽的细胞会导致更多的端粒 DNA 损伤。然而，其根本机制尚不清楚。 同源重组 (HR) 是主要的 VSG 转换途径，但关键 HR 重组酶的删除 RAD51 不会消除重组介导的转换，表明其他重组机制 都参与其中。 T. brucei 中存在微同源介导的末端连接 (MMEJ) 事件，但尚不清楚是否存在 VSG 切换可以通过 MMEJ 发生。抗原变异是一种重要的发病机制 长期寄生虫感染。了解端粒蛋白如何影响 VSG 转换和识别 所有转换途径都将帮助我们开发出在未来根除这种寄生虫的方法。 更好地了解端粒蛋白如何影响 VSG 转换并识别其他重组 涉及抗原变异的机制，我们将研究 TRF 如何帮助维持端粒完整性和 使用新颖的单分子分析——原子力显微镜成像（AFM，在空气和高速条件下）来确定稳定性 液体中）和DNA走钢丝——以及遗传和分子工具，以实现以下目标。在目标 1 中，我们将 通过测试TRF是否直接结合TRL以及TRF是否可以招募来研究TRF如何抑制TRL TERRA 通过不同的 TRF 分子与双链体端粒 DNA 结合，并 同二聚化。我们还将检查 TRF 点突变体中的 TERRA 定位和 R 环水平， 减弱或增强其 TERRA 结合活性。在目标 2 中，我们将利用 TRF 耗尽的细胞 更多重组产物并检查 HR 和 MMEJ 是否有助于端粒/亚端粒 通过删除/敲低 TRF RNAi 细胞中这些途径所必需的因子来稳定不稳定性。然后我们将检查 WT TRF 背景下缺乏关键重组因子的细胞中的 VSG 转换。我们的研究将揭示 TRF 如何维持端粒完整性并识别抗原变异中潜在的其他重要因素。