Structure and function of eukaryotic DNA transposases

真核DNA转座酶的结构和功能

• 批准号: 7734103
• 负责人: Frederick Dyda
• 金额: $ 32.73万
• 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/7734103
• 关键词: Aedes; Amino Acids; Amyotrophic Lateral Sclerosis; Anopheles gambiae; Benign; Cancer Control; Catalytic Domain; Cell physiology; Cells; Characteristics; Chromosomes; Complex; Crystallization; Culicidae; Cystic Fibrosis; DNA; DNA Binding; DNA Transposons; Development; Disease; Disruption; Employee Strikes; Enzymes; Equilibrium; Eukaryotic Cell; Evolution; Excision; Fishes; Foundations; Genes; Genome; Genomics; Goals; Hand; Hemophilia A; Human; Immune system; In Vitro; Insect Control; Insecta; Integrase; Learning; Length; Location; Malaria; Mammalian Cell; Modeling; Moths; Musca domestica; Nature; Nucleic Acid Regulatory Sequences; Pattern; Plants; Proteins; Proteolysis; Relative (related person); Research; Sickle Cell Anemia; Sleeping Beauty; Structure; System; Transgenic Organisms; Transposase; Work; Yellow Fever; gene therapy; interest; member; programs; protein expression; protein folding; recombinase; structural biology; tool; transposon/insertion element; trend; vector

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 recently solved the structure of an N-terminally truncated version of the 612-residue Hermes protein (Hickman et al., 2005). 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 hexameric protein and its complexes with DNA. We can 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 underway. We are also pursuing our interest in DNA transposition systems that function in mammalian cells such as Sleeping Beauty which was resurrected from an inactive version found in salmonid fish (Ivics et al., 1997) and piggyBac, an active moth transposon (Wu et al., 2006; Mitra et al., 2008). 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. Ivics, Z., Hackett, P.B., Plasterk, R.H., and Izsvak, Z. (1997) Cell 91, 501-510. 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.

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。