Polard P, Chandler M
Laboratoire de Microbiologie et Génétique Moléculaires, UPR9007 du Centre National de la Resherche Scientifique (CNRS), Toulouse, France.
Genes Dev. 1995 Nov 15;9(22):2846-58. doi: 10.1101/gad.9.22.2846.
Expression of the bacterial insertion sequence IS911 transposase in vivo leads to excision and circularization of IS911-based transposons. We show here that transposase produces an unusual molecular form generated by single-strand cleavage, transfer, and ligation of one end of the element to the opposite end. When the transposon is carried by a circular plasmid, this results in the formation of a "figure-eight" molecule in which a single strand of the transposon is circularized while the corresponding strand of the vector backbone retains a single-strand interruption at this position. The results show that a 3' end of the transposon is transferred to the opposite target end. Transposase is therefore capable of introducing single-strand cleavages at the ends of the element, an activity similar to that of retroviral integrases with which it shares significant similarities in amino acid sequence. Kinetic studies demonstrate that the figure-eight accumulates earlier than transposon circles after transposase induction and disappears before circles after inhibition of transposase expression, raising the possibility that the figure-eight molecules are precursors to the circles. Therefore, IS911 excision as a circle may not occur by double-strand cleavage leading to its prior separation from the vector backbone in a linear form but could proceed by consecutive circularization of each strand.
细菌插入序列IS911转座酶在体内的表达会导致基于IS911的转座子切除并环化。我们在此表明，转座酶会产生一种不寻常的分子形式，它由元件一端的单链切割、转移并连接到另一端而产生。当转座子由环状质粒携带时，这会导致形成一个“8字形”分子，其中转座子的一条单链环化，而载体骨架的相应链在此位置保留单链中断。结果表明，转座子的一个3'端转移到了相对的靶端。因此，转座酶能够在元件末端引入单链切割，这种活性类似于逆转录病毒整合酶，它与逆转录病毒整合酶在氨基酸序列上有显著相似性。动力学研究表明，在转座酶诱导后，8字形分子比转座子环更早积累，并且在转座酶表达受到抑制后，8字形分子比环更早消失，这增加了8字形分子是环的前体的可能性。因此，IS911以环的形式切除可能不是通过双链切割导致其以线性形式先与载体骨架分离，而是可能通过每条链依次环化来进行。
