Structure and function of eukaryotic DNA transposases

真核DNA转座酶的结构和功能

• 批准号: 10006695
• 负责人: Frederick Dyda
• 金额: $ 87.84万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10006695
• 关键词: Active Sites; Adaptive Immune System; Adopted; Aedes; Affect; Amyotrophic Lateral Sclerosis; Anopheles gambiae; Antibiotic Resistance; Architecture; Bacteria; Binding; Biochemical; Biological Assay; Cancer Control; Cell physiology; Cells; Childhood; Chiroptera; Chromosomes; Cryoelectron Microscopy; Crystallization; Culicidae; Cysteine; Cystic Fibrosis; DNA; DNA Binding; DNA Binding Domain; DNA Sequence Rearrangement; DNA Transposable Elements; DNA Transposons; DNA biosynthesis; Development; Disease; Engineering; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Excision; Family; Foundations; Future; Gene Rearrangement; Genes; Genetic Engineering; Genets; Genome; Genomics; Goals; Growth; Hemophilia A; Histidine; Human; Insecta; Insertional Mutagenesis; Investigation; Ions; Length; Libraries; Location; Malaria; Medical; Molecular; Molecular Conformation; Moths; Musca domestica; Mutate; Mutation; Nature; Nucleic Acids; Oncogenic; Organism; Paste substance; Pathogenesis; Plants; Process; Proteins; Reaction; Research; Rhabdoid Tumor; Sickle Cell Anemia; Site; Stem cells; Structural Biochemistry; Structure; System; Therapeutic; Transact; Transposase; Work; Yellow Fever; Zinc Fingers; biophysical properties; cancer immunotherapy; cell type; chimeric antigen receptor T cells; divalent metal; emerging antibiotic resistance; gene function; gene product; gene therapy; insight; interest; member; novel; programs; promoter; recombinase; reconstitution; repaired; structural biology; tool; transgenic insect; transposon/insertion element; tumor; vector

Eukaryotic DNA transposons can be classified into a number of distinct superfamilies, and one of the most widely distributed of these is the so-called "hAT" superfamily, which has active members in plants and insects. We began our structural studies of eukaryotic DNA transposases (2,3) with Hermes, a hAT transposon that is active not only in the house fly from which it was isolated but also in other insects such as Aedes aegypti, the mosquito species that transmits yellow fever. A close relative of Hermes, the Herves transposon, is active in the malaria vector Anopheles gambiae. An active insect transposon is particularly interesting because it offers the potential to produce transgenic insects for controlling medically significant pests. Hermes transposition employs a mechanism in which excision is accompanied by hairpin formation on the DNA flanking the transposon, as also seen for the RAG1/2 recombinase of the adaptive immune system. We are continuing our investigation into the mechanism of Hermes DNA transposition using both crystallographic and Cryoelectron microscopy approaches. We recently determined several co-crystal structures of Hermes bound to DNA that mimics the reaction step just before hairpin formation (4). These revealed a large DNA conformational change between the initial cleavage step and subsequent hairpin formation. It seems that two factors affect the conformational change: the divalent metal ions bound at the active site, and the identity of a specific flanking basepair. The structures point to the catalytic importance of a histidine residue within a conserved C/DxxH motif present in several other transposase families. Another DNA transposition system of interest to us is piggyBac, an active moth transposon. This transposition system arguably has the widest range of current applications by virtue of its ability to function in many cell types and its "seamless" insertion/excision mechanism that does not require DNA synthesis to repair its sites of action, unique among transposons. Many aspects of its transposition mechanism are not yet well-understood. We have recently shown that the cysteine-rich carboxy-terminal domain of the piggyBac transposase functions as the site-specific DNA binding domain, and have collaborated to solve its structure by NMR (5). We have now successfully expressed and purified the full-length piggyBac transposase, and are in the process of determining its structure when bound to its transposon ends and target DNA. We have also been studying a domesticated piggyBac transposase that is present in most organisms including humans, called piggyBac transposable element derived 5 (PGBD5). Although its function is unclear, the protein has been implicated in genomic rearrangements in childhood rhabdoid tumors (6). We have expressed and purified the PGBD5 protein, and are currently characterizing its biochemical and biophysical properties. We hypothesize that this may provide insight into its cellular function. The third superfamily of eukaryotic DNA transposons that we have been studying are the Helitrons. Although no currently mobile Helitrons have been identified, they must once have been very active, as their remnants are widespread throughout the eukaryotic kingdom. Unlike other known eukaryotic DNA transposons, Helitron insertions in the host genome are not bordered by target site duplications, suggesting a replicative transposition mechanism that differs substantially from the cut-and-paste mode of transposition used by all other currently characterized eukaryotic systems We have been investigating a reconstituted Helitron transposon, Helraiser (7), and have shown that for transposition, the donor site must be double-stranded and that single-stranded donors do not suffice (8). Nevertheless, replication and integration assays reveal that only one of the transposon donor strands is used. Our investigation of Helitron mobility has established the uniqueness of the Helitron transposition mechanism and suggests its potential in future, novel genomic applications. We are extending this work to cryo-electron microscopy studies to understand how it binds and acts on DNA as it carries out transposition. 2. Hickman et al. (2005) Molecular architecture of a eukaryotic DNA transposase. Nature Struct. Mol. Biol. 12, 715-721. 3. Hickman et al. (2014) Structural basis of transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica. Cell 158, 353-367. 4. Hickman et al. (2018) Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase. Nucleic Acids Res. 46, 10286-10301. 5. Morellet et al. (2018) Sequence-specific DNA binding activityof the cross-brace zinc finger motif of the piggyBac transposase. Nucleic Acids Res. 46, 2660-2677. 6. Henssen et al. (2017) PGBD5 promotes site-specific oncogenic mutations in human tumors. Nature Genet. 49, 1005-1014. 7. Grabundzija et al. (2016) A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes. Nature Commun. 7, 10716. 8. Grabundzija, Hickman, and Dyda (2018) Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition. Nature Commun. 9, 1278.

真核DNA转座子可以分为许多不同的超家族，其中分布最广泛的是所谓的"hAT"超家族，其在植物和昆虫中具有活性成员。我们用爱马仕开始了真核DNA转座酶的结构研究（2，3），爱马仕是一种hAT转座子，不仅在从中分离的家蝇中有活性，而且在其他昆虫如传播黄热病的蚊子种类埃及伊蚊中也有活性。爱马仕的近亲Herves转座子在疟疾媒介冈比亚按蚊中具有活性。活性昆虫转座子是特别令人感兴趣的，因为它提供了产生用于控制医学上重要的害虫的转基因昆虫的潜力。爱马仕转座采用了一种机制，其中切除伴随着在转座子侧翼的DNA上形成发夹，这也见于适应性免疫系统的RAG 1/2重组酶。我们正在继续我们的调查机制爱马仕DNA转座使用晶体学和冷冻电子显微镜的方法。我们最近确定了与DNA结合的爱马仕的几种共晶结构，其模拟了发夹形成之前的反应步骤（4）。这些揭示了初始切割步骤和随后的发夹形成之间的大的DNA构象变化。似乎有两个因素影响构象变化：在活性位点结合的二价金属离子，和一个特定的侧翼碱基对的身份。这些结构指出了在其他几个转座酶家族中存在的保守C/DxxH基序内的组氨酸残基的催化重要性。 我们感兴趣的另一个DNA转座系统是piggyBac，一种活跃的蛾转座子。这种转座系统可以说具有最广泛的当前应用，因为它能够在许多细胞类型中发挥作用，并且它的"无缝"插入/切除机制不需要DNA合成来修复其作用位点，这在转座子中是独一无二的。其转座机制的许多方面尚未得到很好的理解。我们最近已经证明piggyBac转座酶的富含半胱氨酸的羧基末端结构域作为位点特异性DNA结合结构域发挥作用，并合作通过NMR解析其结构（5）。我们现在已经成功地表达和纯化了全长piggyBac转座酶，并且正在确定其与转座子末端和靶DNA结合时的结构。 我们还一直在研究一种驯化的piggyBac转座酶，它存在于包括人类在内的大多数生物体中，称为piggyBac转座因子衍生物5（PGBD 5）。虽然其功能尚不清楚，但该蛋白质与儿童横纹肌样肿瘤的基因组重排有关（6）。我们已经表达并纯化了PGBD 5蛋白，目前正在表征其生物化学和生物物理特性。我们假设这可能会提供深入了解其细胞功能。 我们一直在研究的真核DNA转座子的第三个超家族是Helitron。虽然目前没有移动的Helitrons已被确定，他们必须曾经非常活跃，因为他们的残余物是广泛分布在整个真核王国。与其他已知的真核DNA转座子不同，宿主基因组中的Helitron插入不以靶位点重复为边界，这表明复制性转座机制与所有其他目前表征的真核系统所使用的剪切粘贴转座模式有很大不同。我们一直在研究重建的Helitron转座子Helraiser（7），并且已经表明对于转座，供体位点必须是双链的，单链供体是不够的（8）。然而，复制和整合测定揭示仅使用一个转座子供体链。我们对Helitron迁移率的研究建立了Helitron转座机制的独特性，并表明其在未来新的基因组应用中的潜力。我们正在将这项工作扩展到冷冻电子显微镜研究，以了解它如何结合和作用于DNA，因为它进行转座。 2. Hickman等人（2005）真核DNA转座酶的分子结构。自然结构分子12，715 - 721。 3. Hickman等人（2014）转座子末端识别的结构基础由爱马仕，一个八聚体DNA转座酶从家蝇。158号牢房，353 - 367. 4. Hickman等人（2018）对爱马仕（一种hAT家族真核DNA转座酶）形成双链断裂机制的结构见解。Nucleic Acids Res.46，10286 - 10301. 5. Morellet et al.（2018）Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase. Nucleic Acids Res.46，2660 - 2677. 6. Henssen et al.（2017）PGBD5促进人类肿瘤中的位点特异性致癌突变。nature Genet. 49，1005 - 1014。 7. Grazija et al.（2016）从蝙蝠中重建的Helitron转座子揭示了真核生物中基因组改组的新机制。自然通讯7，10716。 8. Grazija，Hickman和Dyda（2018）Helraiser中间体提供了对真核复制转座机制的深入了解。自然通讯9，1278。