Telomere end processing and telomere stability maintenance in trypanosomes

项目摘要 端粒是染色体末端的蛋白质/dna复合体，对保存真核细胞是必不可少的。 增殖性。传统的dna聚合酶不能完全复制线形dna末端，而且大多数真核生物 用端粒酶重新合成富含G的端粒。末端富含G的端粒3‘突起 (G3OH)对于端粒酶介导的G链合成和T环结构的形成是必不可少的，即 对于保护自然染色体末端不受非法DNA过程的影响至关重要。此外，还包括 为了避免过度的端粒重组，需要对G3OH进行调控。端粒的产生 G3OH包括在常规DNA复制后被核酸外切酶切除端粒5‘端，G- 通过端粒酶合成链，并填充C链。在高等真核生物中，这些端粒末端过程是 受动质体寄生虫中不存在的含OB折叠的端粒单链DNA结合蛋白的调节 布氏锥虫。相反，我们已经发现布氏毛滴虫，一种端粒蛋白和一个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。布氏毛滴虫在人类中引起昏睡病，并经常 改变其主要的表面抗原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基因座。我们已经证明，端粒蛋白不仅是 对布氏毛滴虫细胞的增殖至关重要，但也调节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这一事实外，端粒末端的机制 在布氏锥虫中，人们对加工及其调控知之甚少。在这个项目中，我们的目标是调查 布氏支原体端粒末端加工调控及端粒稳定性维持机制。在AIM 1，我们将研究PIRE是如何抑制端粒酶活性的，以及它是否也会影响结局 两条端粒链的核苷酸。布鲁氏毛滴虫的端粒C链填充可能需要DNA 聚合酶。由于PIRE是一种DNA聚合酶，在目标2.1中，我们将测试其DNA聚合酶结构域是否 对于其在端粒末端加工调控中的作用至关重要。我们还鉴定了类似于Primase-Polymerase的 蛋白2(PPL2，具有跨损伤DNA合成活性)作为端粒蛋白，发现PPL2耗竭 显著延长了端粒G3OH的长度。在目标2.2中，我们将调查PPL2是否对 端粒C链填充以及这一功能是否依赖于脊髓。布鲁氏毛滴虫没有非- 同源末端连接(NHEJ)机制，而功能障碍的端粒经常通过NHEJ融合在一起 高等真核生物。在目标3中，我们将研究同源重组和微同源- 介导的末端连接是WT和脊髓灰质炎耗竭细胞端粒重组的主要途径，这将 有助于揭示威胁布氏毛滴虫自然染色体末端的非法过程。我们的研究将对我们有所帮助 我们更好地了解端粒蛋白的进化，为未来抗寄生虫药物的开发做出贡献。

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.