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)。其中分布最广泛的是HAT转座子超家族，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DNA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转座在体外被重复，并通过一种机制进行，其中切除伴随着在转座子两侧的DNA上形成发夹(周等人，2004年)，就像适应性免疫系统的RAG1/2重组酶一样。我们最近解决了612个残基的Hermes蛋白的N-末端截短版本的结构。蛋白质折叠显示了一个围绕DDE转座酶的逆转录病毒整合酶样催化核心的四个结构域的蛋白质。DDE催化核心被一个大的插入结构域破坏，其结构与迄今鉴定的任何其他蛋白质不同，它的存在符合以下趋势，即能够在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发夹所需的氨基酸。另一方面，在原核生物转座酶中看到的范例之一-它们在DNA结合时组装成活性多聚体复合体-没有被遵循。奇怪的是，爱马仕被预先组装成一个六角体。由于我们只结晶了一种蛋白质降解的二聚体Hermes，我们利用我们的结构结果提出了一个六角体结构的模型；这个模型基于一个亚基环，其中环周围的相邻亚基对代表催化活性单元。 我们目前的重点是全长蛋白质及其与DNA的复合体。最近，我们已经能够设计出表达和纯化全长Hermes的条件，产量适合结晶研究。Hermes与转座子末端序列的复合体是单分散的，高度可溶，目前正在进行结晶试验。我们已经开始研究埃及伊蚊和卡氏赤霉菌相关的HAT转座酶，它们可能具有更容易与DNA共结晶的生物物理性质。 我们还对目前用于哺乳动物细胞研究的DNA转座系统感兴趣，包括复活的睡美人转座子(IVICS等人，1997年)和PiggyBac(Wu等人，2006年)。由于这些转座子的应用受到转座子只能在某些细胞类型中转座的能力和非特异性靶向的限制，我们相信真核转座酶的X射线结构可以为研究其机制和调控提供重要的见解。 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。 艾维克斯，Z.，哈克特，P.B.，普拉斯特克，R.H.和伊兹瓦克，Z.(1997)细胞91,501-510。 吴绍中，梅尔，Y.J.，Coates，C.J.，Handler，A.M.，Pelczar，P.，Moisyadi，S.，and Kaminski，J.M.(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。 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。 周利勤，Mitra R.，Atkinson P.W.，Hickman A.B.，Dyda F.，Craig N.L.(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 hAT transposon superfamily, which has active members in plants and insects. We began our structural studies of eukaryotic DNA transposases with Hermes, a DNA transposon that is active in not only 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 proceeds through 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 recently solved the structure of an N-terminally truncated version of the 612-residue Hermes protein. The protein fold revealed a four-domain 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 structure does not resemble any other protein characterized to date, and its 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 paradigms 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. As we crystallized only a proteolyzed dimeric form of Hermes, we used our structural results to propose a model for the structure of the hexamer; this model is based on a ring of subunits in which neighboring pairs of subunits around the ring represent the catalytically active unit. Our current focus is the full-length protein and its complexes with DNA. We have recently been able to work out conditions to express and purify full-length Hermes with yields suitable for crystallization studies. Complexes of Hermes with transposon end sequences are monodisperse and highly soluble, and crystallization trials are currently underway. We have initiated studies on related hAT transposases from Aedes aegypti and Tribolium castaneum which may have biophysical properties more amenable to co-crystallization with DNA. We are also interested in DNA transposition systems in current use in the study of mammalian cells, including the resurrected Sleeping Beauty transposon (Ivics et al., 1997) and piggyBac (Wu et al., 2006). As the applications of these transposons can be limited by the ability to transpose in only certain cell types and non-specific targeting, we believe that X-ray structures of eukaryotic transposases can provide important insights into aspects of their mechanisms and regulation. Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G., and Jenkins, N.A. (2005) Nature 436, 221-226. Ivics, Z., Hackett, P.B., Plasterk, R.H., and Izsvak, Z. (1997) Cell 91, 501-510. Wu, S.C., Meir, Y.J., Coates, C.J., Handler, A.M., Pelczar, P., Moisyadi, S., and Kaminski, J.M. (2006) Proc. Natl. Acad. Sci. USA 103, 15008-15013. Kapitonov, V.V. and Jurka, J. (2004) DNA Cell Biol. 23, 311-324. 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., Mitra R., Atkinson P.W., Hickman A.B., Dyda F., and Craig N.L. (2004) Nature 432, 995-1001.