Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity

布氏锥虫 TRF 维持端粒完整性的机制

基本信息

批准号：
10526882
负责人：
Bibo Li
金额：
$ 23.27万
依托单位：
CLEVELAND STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-16 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10526882
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

项目摘要

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.

项目摘要/摘要 Brucei锥虫，Cruzi和Leishmania是密切相关的动质寄生虫导致人类疾病使人衰弱。 T. Brucei顺序表达免疫学上不同的变体表面糖蛋白（VSGS），其主要的表面抗原，仅来自亚电体VSG表达位点（ESS）逃避宿主免疫反应。同样，发生抗原的许多其他真核病原体变异还表达其主要的表面抗原来自亚teloseres，而DNA重组是一个重要的抗原切换的手段。布鲁氏菌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切换，同时丢失活性VSG附近的基因完整性导致几乎90％的细胞致死率。我们已经证明了TRF，双链端粒DNA结合因子在维持端粒完整性和稳定性方面起着重要作用。这 RNA聚合酶I转录到Terra中，活性VSG-ADJACENT端粒可转录到Terra中，可以形成远程测量带有远程DNA的R环（TRL）。 TRF抑制Terra和TRL水平，以及TRF-的更多TRL 耗尽的细胞会导致更远程的DNA损伤。但是，基本机制尚不清楚。同源重组（HR）是主要的VSG开关途径，但要删除关键HR重组酶 RAD51不会消除重组介导的切换，表明其他重组机制参与。 Brucei中存在微型学介导的结束（MMEJ）事件（MMEJ）事件，但尚不清楚是否是否知道 VSG切换可以通过MMEJ发生。抗原变异是一种必不可少的发病机制长期寄生虫感染。了解端粒蛋白如何影响VSG切换和识别所有切换途径将有助于我们开发未来的寄生虫的方法。更好地了解端粒蛋白如何影响VSG切换并识别其他重组参与抗原变异的机制，我们将研究TRF如何帮助维持端粒完整性和使用新型的单分子分析 - 原子力显微镜成像（AFM，空气和高速）稳定性在AIM 1中，我们将检查TRF如何通过测试TRF是否直接绑定TRL以及TRF是否可以募集来抑制TRL 通过通过不同的TRF分子与两个核酸结合，到双链远程远程远程DNA和同构化。我们还将检查TRF点突变体中的Terra定位和R环水平弱或增强其Terra结合活性。在AIM 2中，我们将利用TRF耗尽的细胞具有更多的重组产品并检查HR和MMEJ是否有助于远程远程/亚电体通过删除/敲低因子对TRF RNAi细胞中这些途径必不可少的不稳定性。然后我们将检查 VSG在WT TRF背景中缺乏关键重组参与者的单元格中切换。我们的研究将揭示 TRF如何保持端粒完整性并确定抗原变异中潜在的其他重要因素。