Structure and function of eukaryotic DNA transposases

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，它在植物和昆虫中都有活跃的成员。我们用Hermes开始了对真核DNA转座酶(2，3)的结构研究，Hermes是一种帽子转座子，不仅在分离它的家蝇中活跃，而且在其他昆虫中也活跃，如埃及伊蚊，传播黄热病的蚊子物种。Herves转座子是Hermes的近亲，在疟疾媒介冈比亚按蚊中很活跃。活跃的昆虫转座子特别有趣，因为它提供了产生转基因昆虫的潜力，以控制具有重大医学意义的害虫。Hermes转座采用了一种机制，即切除伴随着在转座子两侧的DNA上形成发夹，就像适应性免疫系统的RAG1/2重组酶一样。我们正在继续使用结晶学和低温电子显微镜方法研究Hermes DNA转座的机制。我们最近确定了几个与DNA结合的Hermes的共晶结构，这些结构模仿了发夹形成之前的反应步骤(4)。这表明在最初的切割步骤和随后的发夹形成之间有很大的DNA构象变化。似乎有两个因素影响构象变化：结合在活性中心的二价金属离子，以及特定侧翼碱基的身份。这些结构表明，在其他几个转座酶家族中存在的保守C/DxxH基序中，组氨酸残基具有催化作用。 我们感兴趣的另一个DNA转座系统是一种活跃的飞蛾转座子--PiggyBac。可以说，这种转座系统具有目前最广泛的应用范围，因为它能够在多种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合成来修复其作用部位，这在转座子中是独一无二的。它的转位机制的许多方面还没有被很好地理解。我们最近证明了iggyBac转座酶的富含半胱氨酸的羧基末端结构域起到了位点特异性DNA结合域的作用，并通过核磁共振(5)合作求解了它的结构。我们现在已经成功地表达和纯化了全长的iggyBac转座酶，并正在确定它与转座子末端和靶DNA结合时的结构。 我们也一直在研究一种驯化的猪Bac转座酶，它存在于包括人类在内的大多数生物体中，称为猪Bac转座元件衍生5(PGBD5)。尽管其功能尚不清楚，但该蛋白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PGBD5蛋白，目前正在对其生化和生物物理性质进行鉴定。我们推测，这可能会提供对其细胞功能的洞察。 我们一直在研究的第三个真核DNA转座子超家族是Helitrons。尽管目前还没有发现可移动的Helitrons，但它们一定曾经非常活跃，因为它们的残留物在整个真核生物王国中广泛存在。与其他已知的真核DNA转座子不同，在宿主基因组中插入Helitron不会受到靶点复制的限制，这表明了一种复制转座机制，它与所有其他目前具有特征的真核系统所使用的剪切粘贴转座模式有很大不同。我们一直在研究一种重组的Helriser转座子(7)，并表明对于转座，供体部位必须是双链的，单链供体是不够的。然而，复制和整合分析表明只使用了转座子供体链中的一个。我们对氦电子迁移率的研究已经确立了氦电子转座机制的独特性，并暗示了它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，以了解它在进行转座时如何结合和作用于DNA。 2.希克曼等人。(2005)真核DNA转座酶的分子结构。大自然的结构。摩尔。比奥尔。12,715-721。 3.希克曼等人。(2014)Hermes转座子末端识别的结构基础，Hermes是一种来自家蝇的八聚体DNA转座酶。158号，353-367号。 4.希克曼等人。(2018)HAT家族真核DNA转座酶Hermes双链断裂形成机制的结构洞察。核酸研究报告46,10286-10301。 5.Morellet等人。(2018)iggyBac转座酶的交叉支撑锌指基序的序列特异性DNA结合活性。核酸决议46,2660-2677。 6.Henssen等人。(2017)PGBD5促进人类肿瘤中的部位特异性致癌基因突变。大自然的吉内特。49,1005-1014。 7.Grabundzija等人。(2016)从蝙蝠中重建的氦电子转座子揭示了真核生物基因组改组的一种新机制。自然界的普通人。7,10716。 8.Grabundzija，Hickman和Dyda(2018)Helriser中间体提供了对真核复制转座机制的洞察。自然界的普通人。9,1278。

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.