Structure and function of eukaryotic DNA transposases

真核DNA转座子可分为十个左右的超家族(Kapitonov&Jurka，2004)。分布最广的是所谓的“帽子”超家族，它在植物和昆虫中都有活跃的成员。我们从Hermes开始了对真核DNA转座酶的结构研究，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(Sarkar等人，1997年)，这是一种传播黄热病的蚊子物种。Herves转座子是Hermes的近亲，活跃在疟疾媒介冈比亚按蚊中(Arensburger等人，2005年)。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转座已在体外被概括，并被证明采用了一种机制，即切除伴随着转座子两侧DNA上的发夹形成(周等人，2004年)，就像适应性免疫系统的RAG1/2重组酶一样。我们已经确定了612个残基的Hermes蛋白的N-末端截短版本的结构(Hickman等人，2005年)，蛋白质折叠揭示了围绕DDE转座酶的逆转录病毒整合酶样催化核心组织的多域蛋白。DDE催化核心被一个大的插入结构域破坏，该插入结构域的存在符合以下趋势：能够在DNA底物上形成发夹的DDE转座酶需要一个辅助结构域来提供促进发夹形成和稳定它们所需的氨基酸。另一方面，原核生物转座酶中的一种模式--它们在DNA结合时组装成活性多聚体复合体--没有得到遵循。奇怪的是，爱马仕被预先组装成一个六角体。在我们最初的研究中，我们只结晶了一种蛋白质水解型的Hermes二聚体，并利用我们的结构结果提出了一个六聚体结构的模型。 我们目前的重点是全长蛋白质及其与DNA的复合体。我们可以表达和纯化全长Hermes，产量适合结晶研究。金属离子分析证实，该蛋白质与一个锌离子结合，与N-末端床区的存在一致，该结构域可能在非特异性DNA结合或蛋白质-蛋白质相互作用中发挥作用。Hermes与转座子末端序列的复合体是单分散的，高度可溶，结晶试验正在进行中。 我们还对在哺乳动物细胞中发挥作用的DNA转座系统感兴趣，例如青冈鱼的Tol2和一种活跃的飞蛾转座子iggyBac(Wu等人，2006年；Mitra等人，2008年)。Tol2也是一种表达良好且易于纯化的蛋白质，这种HAT转座酶的结晶试验也在进行中。为了研究真核转座酶可能经历对活性很重要的翻译后修饰的可能性，我们还在研究在真核细胞系中表达的蛋白质。 杜佩，A.J.，赤木，K.，拉格斯帕达，D.A.，科普兰，N.G.和詹金斯，N.A.(2005)自然，436,221-226。 希克曼，A.B.等人。(2005)NAT。结构。摩尔。比奥尔。12,715-721。 Wu，S.C.等人。(2006)Proc.娜塔莉。阿卡德。SCI。美国第103,15008-15013。 王晓明，等.DNA细胞生物学(2004).23,311-324。 米特拉，R.，Fain-Thornton，J.和Craig，N.L.(2008)EMBO J.27,1097-1109。 Sarkar，A.，Yardley，K.，Atkinson，P.W.，James，A.A.和O‘Brochta，D.A.(1997)昆虫生物化学。摩尔。比奥尔。27,359-363。 周利清，等人。(2004)自然432,995-1001。

Eukaryotic DNA transposons can be classified into ten or so superfamilies (Kapitonov & Jurka, 2004). One of the most widely distributed is the so-called "hAT" superfamily, which has active members in plants and insects. We began our structural studies of eukaryotic DNA transposases 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 (Sarkar et al., 1997), the mosquito species that transmits yellow fever. A close relative of Hermes, the Herves transposon, is active in the malaria vector Anopheles gambiae (Arensburger et al., 2005). An active insect transposon is particularly interesting because it offers the potential to produce transgenic insects for controlling medically significant pests. Hermes transposition has been recapitulated in vitro and shown to employ a mechanism in which excision is accompanied by hairpin formation on the DNA flanking the transposon (Zhou et al., 2004), as also seen for the RAG1/2 recombinase of the adaptive immune system. We have determined the structure of an N-terminally truncated version of the 612-residue Hermes protein (Hickman et al., 2005), and the protein fold revealed a multidomain protein organized around the retroviral integrase-like catalytic core characteristic of DDE transposases. The DDE catalytic core is disrupted by a large insertion domain whose presence conforms to the trend that DDE transposases capable of forming hairpins on their DNA substrates require an ancillary domain to provide the amino acids needed to promote hairpin formation and to stabilize them. On the other hand, one of the patterns seen in prokaryotic transposases - that they assemble into an active multimeric complex upon DNA binding - is not followed. Curiously, Hermes is pre-assembled as a hexamer. In our initial studies, we crystallized only a proteolyzed dimeric form of Hermes, and we used our structural results to propose a model for the structure of the hexamer. Our current focus is the full-length protein and its complexes with DNA. We can express and purify full-length Hermes with yields suitable for crystallization studies. Metal ion analysis confirmed that the protein binds one zinc ion, consistent with the presence of an N-terminal BED domain that may play a role in either non-specific DNA binding or protein-protein interactions. Complexes of Hermes with transposon end sequences are monodisperse and highly soluble, and crystallization trials are underway. We are also pursuing our interest in DNA transposition systems that function in mammalian cells such as Tol2 from the medaka fish and piggyBac, an active moth transposon (Wu et al., 2006; Mitra et al., 2008). Tol2 is also a well expressed and readily purifiable protein, and crystallization trials are also underway with this hAT transposase. To examine the possibility that eukaryotic transposases may undergo posttranslational modifications that are important for activity, we are also studying the proteins when expressed in eukaryotic cell lines. Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G., and Jenkins, N.A. (2005) Nature 436, 221-226. Hickman, A.B., et al. (2005) Nat. Struct. Mol. Biol. 12, 715-721. Wu, S.C., et al. (2006) Proc. Natl. Acad. Sci. USA 103, 15008-15013. Kapitonov, V.V. and Jurka, J. (2004) DNA Cell Biol. 23, 311-324. Mitra, R., Fain-Thornton, J., and Craig, N.L. (2008) EMBO J. 27, 1097-1109. Sarkar, A., Yardley, K., Atkinson, P.W., James, A.A., and O'Brochta, D.A. (1997) Insect Biochem. Mol. Biol. 27, 359-363. Zhou L.Q., et al. (2004) Nature 432, 995-1001.