Telomere end processing and telomere stability maintenance in trypanosomes

锥虫的端粒末端加工和端粒稳定性维持

• 批准号: 10677878
• 负责人: Bibo Li
• 金额: $ 29.7万
• 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: 2022-08-05 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677878
• 关键词: Affect; African Trypanosomiasis; Antigenic Variation; Binding; Binding Proteins; Biological Assay; Biology; Cell Proliferation; Cells; Chromosomes; Complex; DNA; DNA Primase; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA polymerase alpha-primase; DNA-Directed DNA Polymerase; Development; EXO1 gene; Ensure; Eukaryota; Eukaryotic Cell; Evolution; Excision; Exonuclease; Family; Future; Generations; Genetic Recombination; Goals; Human; Immune Evasion; Immune response; Infection; Knowledge; Label; Length; Ligation; Maintenance; Mediating; Nonhomologous DNA End Joining; Nucleotides; Oligonucleotides; Organism; Parasites; Pathogenesis; Pathway interactions; Play; Polymerase; Process; Proliferating; Proteins; Regulation; Resected; Role; Single-Stranded DNA; Structure; Surface Antigens; Telomerase; Telomere Maintenance; Testing; Trypanosoma; Trypanosoma brucei brucei; genome integrity; homologous recombination; improved; mutant; novel; nuclease; recruit; telomere

Project Summary Telomere, the protein/DNA complex at the chromosome end, is essential for keeping eukaryotic cells proliferative. Conventional DNA polymerases cannot fully replicate the linear DNA ends, and most eukaryotes use telomerase to synthesize the telomere G-rich strand de novo. The terminal telomere G-rich 3’ overhang (G3OH) is essential for telomerase-mediated G-strand synthesis and formation of the T-loop structure that is critical for protection of the natural chromosome ends from illegitimate DNA processes. In addition, the length of G3OH needs to be regulated in order to avoid excessive telomeric recombination. Generation of the telomere G3OH involves resection of the telomere 5’ end by exonucleases after the conventional DNA replication, G- strand synthesis by telomerase, and C-strand filled-in. In higher eukaryotes, these telomere end processes are regulated by OB fold-containing telomere ssDNA-binding proteins, which are absent in the kinetoplastid parasite Trypanosoma brucei. Rather, we have found that T. brucei PolIE, a telomere protein and an A family DNA polymerase, suppresses telomerase-mediated G-strand extension, helps ensure proper telomere C-strand synthesis, and suppresses telomeric recombination. T. brucei causes sleeping sickness in humans and regularly switches its major surface antigen, VSG, to achieve immune evasion. VSGs are monoallelically expressed exclusively from loci immediately upstream of the telomere. We have shown that telomere proteins not only are essential for T. brucei cell proliferation but also regulate VSG monoallelic expression and switching. However, other than the fact that telomerase can synthesize the telomere G-strand DNA, mechanisms of telomere end processing and its regulation are poorly understood in T. brucei. In this project, we aim to investigate mechanisms of T. brucei telomere end processing regulation and telomere stability maintenance. In Aim 1, we will investigate how PolIE suppresses the telomerase action and whether it also affects the ending nucleotides of the two telomere strands. The telomere C-strand fill-in in T. brucei presumably requires a DNA polymerase. Since PolIE is a DNA polymerase, in Aim 2.1, we will test whether its DNA polymerase domain is critical for its role in telomere end processing regulation. We have also identified Primase-Polymerase-like protein 2 (PPL2, with a translesion DNA synthesis activity) as a telomere protein and found that PPL2 depletion dramatically elongates the telomere G3OH length. In Aim 2.2, we will investigate whether PPL2 is important for telomere C-strand fill-in and whether this function is PolIE-dependent. T. brucei does not have the non- homologous end joining (NHEJ) machinery, while dysfunctional telomeres are frequently fused through NHEJ in higher eukaryotes. In Aim 3, we will investigate whether homologous recombination and Microhomology- mediated end joining are major pathways for telomeric recombination in WT and PolIE-depleted cells, which will help reveal illegitimate processes that threaten the natural chromosome ends in T. brucei. Our studies will help us better understand the telomere protein evolution and contribute to future development of anti-parasite agents.

项目概要 端粒是染色体末端的蛋白质/DNA 复合物，对于维持真核细胞至关重要 增殖的。传统的 DNA 聚合酶无法完全复制线性 DNA 末端，并且大多数真核生物 使用端粒酶从头合成富含 G 的端粒链。富含 G 的末端端粒 3' 突出 (G3OH) 对于端粒酶介导的 G 链合成和 T 环结构的形成至关重要 对于保护天然染色体末端免受非法 DNA 加工至关重要。另外，长度为 G3OH 需要进行调节以避免过度的端粒重组。端粒的产生 G3OH 涉及在常规 DNA 复制后通过核酸外切酶切除端粒 5' 端，G- 端粒酶链合成，C链填充。在高等真核生物中，这些端粒末端过程是 受包含 OB 折叠的端粒 ssDNA 结合蛋白调节，动质体寄生虫中不存在这种蛋白 布氏锥虫。相反，我们发现 T. brucei PolIE、一种端粒蛋白和 A 家族 DNA 聚合酶，抑制端粒酶介导的 G 链延伸，有助于确保正确的端粒 C 链 合成，并抑制端粒重组。布氏锥虫经常引起人类昏睡病 转换其主要表面抗原 VSG，以实现免疫逃避。 VSGs是单等位基因表达的 完全来自端粒上游的位点。我们已经证明端粒蛋白不仅 对于 T. brucei 细胞增殖至关重要，而且还调节 VSG 单等位基因的表达和转换。然而， 除了端粒酶可以合成端粒G链DNA这一事实之外，端粒末端的机制 人们对布氏锥虫的加工及其调控知之甚少。在这个项目中，我们的目标是调查 布氏锥虫端粒末端加工调节和端粒稳定性维持的机制。瞄准 1、我们将研究PolIE如何抑制端粒酶的作用以及是否也影响结局 两条端粒链的核苷酸。 T. brucei 中的端粒 C 链填充可能需要 DNA 聚合酶。由于PolIE是一种DNA聚合酶，因此在Aim 2.1中，我们将测试其DNA聚合酶结构域是否为 对其在端粒末端加工调节中的作用至关重要。我们还鉴定了类似引物酶聚合酶 蛋白2（PPL2，具有跨损伤DNA合成活性）作为端粒蛋白，发现PPL2耗尽 显着延长端粒 G3OH 长度。在目标 2.2 中，我们将研究 PPL2 对于 端粒 C 链填充以及此功能是否依赖于 PolIE。 T. brucei 不具有非 同源末端连接（NHEJ）机制，而功能失调的端粒经常通过 NHEJ 融合 高等真核生物。在目标 3 中，我们将研究同源重组和微同源是否- 介导的末端连接是 WT 和 PolIE 耗尽细胞中端粒重组的主要途径，这将 帮助揭示威胁布氏锥虫天然染色体末端的非法过程。我们的研究将有所帮助 我们更好地了解端粒蛋白的进化，并为未来抗寄生虫药物的开发做出贡献。