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

基本信息

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
项目类别：
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

项目摘要

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传统的 DNA 聚合酶不能完全复制线性 DNA 末端，并且大多数真核生物都具有增殖能力。使用端粒酶从头合成端粒富含 G 的链末端端粒富含 G 的 3’ 突出端。 (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 加工的影响至关重要。需要调节 G3OH 以避免端粒的过度重组。 G3OH 涉及在常规 DNA 复制后通过核酸外切酶切除端粒 5' 端，G- 在高等真核生物中，这些端粒末端过程是由端粒酶合成的。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相反，我们发现了布氏锥虫 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。布氏锥虫经常导致人类昏睡病。改变其主要表面抗原 VSG，以实现免疫逃避 VSG 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是对于 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。除了端粒酶可以合成端粒G链DNA这一事实之外，端粒末端的机制我们对布氏菌的加工及其调控知之甚少。在这个项目中，我们的目的是进行调查。布氏锥虫端粒末端加工调节和端粒稳定性维持的机制。 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可能需要 DNA 来填补 T. brucei 中的端粒 C 链。由于PolIE是一种DNA聚合酶，因此在目标2.1中，我们将测试其DNA聚合酶结构域是否为DNA聚合酶。我们还发现了其在端粒末端加工调节中的关键作用。蛋白2（PPL2，具有跨损伤DNA合成活性）作为端粒蛋白，发现PPL2耗尽在目标 2.2 中，我们将研究 PPL2 对于端粒 G3OH 长度是否重要。端粒C链填充以及该功能是否依赖于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 融合在目标 3 中，我们将研究高等真核生物是否存在同源重组和微同源性。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 T. brucei 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。