Mitra Rupak, Fain-Thornton Jennifer, Craig Nancy L
Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA.
EMBO J. 2008 Apr 9;27(7):1097-109. doi: 10.1038/emboj.2008.41. Epub 2008 Mar 20.
DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3'OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases.
DNA合成被认为是转座元件移动的一个决定性特征。在确定piggyBac转座机制时，piggyBac是一种昆虫转座子，越来越多地用于包括哺乳动物细胞在内的各种系统中的基因组操作，我们发现，在piggyBac转座过程中，无论是在转座子切除后的供体位点，还是在转座子整合后的插入位点，都可以避免DNA合成。我们证明，piggyBac转座子切除是通过在转座子末端形成瞬时发夹结构发生的，并且piggyBac靶标连接是通过转座子3'OH末端直接攻击靶标DNA发生的。这与转座酶和逆转录病毒整合酶的DDE超家族成员用于靶标连接的策略相同。使用我们在酿酒酵母中建立的piggyBac转座系统对突变的piggyBac转座酶进行体外和体内分析表明，piggyBac转座酶是重组酶DDE超家族的成员，这是一个意想不到的结果，因为piggyBac与重组酶DDE家族之间缺乏序列相似性。
