Mechanisms of how Trypanosoma brucei TRF maintains telomere integrity

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.布氏杆菌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，仅来自亚端粒VSG表达位点（ES） 来逃避宿主的免疫反应类似地，许多其他真核病原体， 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。T.布鲁氏菌端粒生物学已经表明，端粒扰动 结构可能是一把双刃剑：增加端粒的稳定性抑制了VSG转换， 活性VSG附近的基因完整性导致近90%的细胞致死率。我们已经表明， 双链端粒DNA结合因子，在维持端粒的完整性和稳定性中起重要作用。的 活性的VSG-邻近端粒被RNA聚合酶I转录成TERRA，其可以形成端粒 端粒DNA的R环（TRL）。TRF抑制TERRA和TRL水平，TRF中的TRL更多- 耗尽的细胞会导致更多的端粒DNA损伤。然而，其潜在机制尚不清楚。 同源重组（HR）是VSG转换的主要途径，但关键HR重组酶的缺失 RAD51不能消除重组介导的转换，表明其他重组机制 参与其中微同源介导的末端连接（MMEJ）事件在T。布氏杆菌，但不知道是否 VSG切换可以通过MMEJ发生。抗原变异是一种重要的致病机制， 长期寄生虫感染了解端粒蛋白如何影响VSG转换和鉴定 所有的转换途径将有助于我们开发出在未来根除这种寄生虫的手段。 为了更好地了解端粒蛋白如何影响VSG转换，并确定额外的重组 我们将研究TRF如何帮助维持端粒的完整性， 使用新型单分子分析的稳定性-原子力显微镜成像（AFM，在空气中和高速 液体）和DNA tightropes-和遗传和分子工具，通过以下目标。在目标1中，我们 通过测试TRF是否直接与TRL结合以及TRF是否可以招募 TERRA通过不同的TRF分子与两种核酸结合， 同源二聚化。我们还将研究TRF点突变体的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耗尽的细胞具有 更多的重组产物，并检查HR和MMEJ是否有助于端粒/亚端粒 通过在TRF RNAi细胞中删除/敲低这些途径所必需的因子来实现不稳定性。然后我们将检查 在WT TRF背景中缺乏关键重组参与者的细胞中的VSG切换。我们的研究将揭示 TRF如何维持端粒的完整性和识别抗原变异中潜在的其他重要因素。

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.