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: 10503111
• 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/10503111
• 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; GTP-Binding Protein alpha Subunits, Gs; 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; Proteins; Regulation; Role; 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 端粒是位于染色体末端的蛋白质/DNA复合物， 增殖传统的DNA聚合酶不能完全复制线性DNA末端，大多数真核生物 用端粒酶重新合成端粒富含G的链。端粒末端富含G的3'突出端 （G3 OH）对于端粒酶介导的G链合成和T环结构的形成是必需的， 对于保护天然染色体末端免受非法DNA过程的影响至关重要。此外， G3 OH需要被调节以避免过度的端粒重组。端粒的产生 G3 OH涉及在常规DNA复制后通过核酸外切酶切除端粒5'末端，G- 通过端粒酶进行链合成，以及C-链填充。在高等真核生物中，这些端粒末端突起是 受含OB折叠的端粒ssDNA结合蛋白调节，这在动质体寄生虫中不存在 布氏锥虫相反，我们发现T.布氏杆菌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，有助于确保适当的端粒C链 合成，并抑制端粒重组。T.布鲁氏杆菌会引起人类昏睡病， 转换其主要表面抗原VSG以实现免疫逃避。VSG单等位基因表达 仅来自紧邻端粒上游的基因座。我们已经证明，端粒蛋白不仅是 对T.布氏细胞增殖，而且还调节VSG单等位基因表达和转换。然而，在这方面， 除了端粒酶可以合成端粒G链DNA外， 对T.布鲁塞。在这个项目中，我们的目标是调查 T.布鲁氏菌端粒末端加工调控和端粒稳定性维持。在Aim中 1、研究PolIE如何抑制端粒酶活性，是否也影响端粒酶活性的终止， 两条端粒链的核苷酸。T.布氏杆菌可能需要DNA 聚合酶。由于PolIE是一种DNA聚合酶，在Aim 2.1中，我们将测试其DNA聚合酶结构域是否 其在端粒末端加工调节中的作用至关重要。我们还发现了类似引物聚合酶的 蛋白2（PPL 2，具有跨损伤DNA合成活性）作为端粒蛋白，并发现PPL 2缺失 显著延长端粒G3 OH长度。在目标2.2中，我们将研究PPL 2是否对 端粒C-链填充以及该功能是否是PolIE依赖性的。T.布鲁塞没有非- 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性是否- 介导的末端连接是WT和PolIE缺失细胞中端粒重组的主要途径， 有助于揭示威胁T.布鲁塞。我们的研究将有助于 我们更好地了解端粒蛋白的进化，并有助于未来的抗寄生虫药物的开发。