Structure and function of eukaryotic DNA transposases

真核生物DNA转座子可分为10个左右的超家族（Kapitonov & Jurka, 2004）。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。我们开始了真核生物DNA转座子Hermes的结构研究，Hermes是一种hAT转座子，它不仅在家蝇中有活性，而且在其他昆虫中也有活性，如埃及伊蚊（Sarkar et al., 1997），这是一种传播黄热病的蚊子。Hermes的近亲Herves转座子在疟疾媒介冈比亚按蚊（Anopheles gambiae）中很活跃（Arensburger等，2005）。一个活跃的昆虫转座子特别有趣，因为它提供了生产转基因昆虫来控制医学上重要的害虫的潜力。

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.