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.

项目摘要 端粒是染色体末端的蛋白质/DNA复合物，对于保持真核细胞至关重要 增殖。常规的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Novo的端粒G-Rich Strand de。端子端粒G-RICH 3'悬垂 （G3OH）对于端粒酶介导的G链合成和T-Loop结构的形成至关重要 保护自然染色体的至关重要的至关重要的DNA过程。另外，长度 需要调节G3OH，以避免过量的远程重组。产生端粒 G3OH涉及在常规DNA复制后通过外核酸切除端粒5'的切除。 端粒酶的链合成，并填充C链。在较高的真核生物中，这些端粒最终过程是 由含有OB折叠的端粒结合蛋白调节，这些结合蛋白不存在于动型寄生虫中 锥虫锥虫。相反，我们发现T. Brucei Polie，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 合成并抑制远程重组。 T. Brucei会导致人类熟睡 切换其主要的表面抗原VSG，以实现免疫进化。 VSG是单相表达的 仅来自端粒上游的基因座。我们已经表明端粒蛋白不仅是 对于布鲁氏菌细胞增殖必不可少的，但也调节VSG单相表达和切换。然而， 除了端粒可以合成端粒G链DNA的事实，端粒端的机制 在T. Brucei中，处理及其调节知之甚少。在这个项目中，我们旨在调查 布鲁氏菌端粒处理调节和端粒稳定性维护的机制。目标 1，我们将调查polie如何抑制端粒酶的作用以及它是否也影响结局 两个端粒链的核苷酸。端粒c链填充在t。brucei中可能需要DNA 聚合酶。由于polie是DNA聚合酶，在AIM 2.1中，我们将测试其DNA聚合酶结构域是否为 它在端粒末端处理调节中的作用至关重要。我们还鉴定了类似于原始酶 - 聚合酶样 蛋白2（ppl2，具有跨性DNA合成活性）作为端粒蛋白，发现PPL2部署 动态拉长端粒G3OH长度。在AIM 2.2中，我们将调查PPL2是否对 端粒C链填充以及此功能是否依赖于polie。 T. Brucei没有非 同源端连接（NHEJ）机械，而功能失调的端粒经常通过NHEJ融合 较高的真核生物。在AIM 3中，我们将研究是否同源重组和微学学 - 介导的末端连接是WT和贫血细胞中远程重组的主要途径，该途径将 帮助揭示威胁天然染色体在T. Brucei结束的非法过程。我们的研究将有所帮助 我们更好地了解端粒蛋白的进化，并有助于抗寄生虫的未来发展。