Drosophila P-element transposase is a novel site-specific endonuclease
Drosophila P-element transposase is a novel site-specific endonuclease
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DOI：
10.1101/gad.11.16.2137
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发表时间：
1997-08-15
影响因子：
10.5
通讯作者：
Rio, DC
中科院分区：
文献类型：
--
作者：
Beall, EL;Rio, DC
我们开发了体外测定法来研究 P 元件转座反应的第一步：供体 DNA 切割。我们发现 P 元件转座酶需要 5' 和 3' P 元件末端才能进行有效的 DNA 切割，这表明突触复合物在切割之前形成。转座酶在 P 元件末端进行交错切割，这对于所有已知的位点特异性核酸内切酶来说都是新颖的：3' 切割位点位于 P 元件的末端，而 5' 切割位点位于 P 元件 31 bp 反向重复序列内的 17 bp。 P 元件末端在裂解反应后免受核酸外切降解，表明裂解后稳定的蛋白质复合物仍与元件末端结合。这些数据与 P 元件转位的剪切和粘贴机制一致，并且可以解释为什么 P 元件主要在体内不精确地切除。
We developed in vitro assays to study the first step of the P-element transposition reaction: donor DNA cleavage. We found that P-element transposase required both 5' and 3' P-element termini for efficient DNA cleavage to occur, suggesting that a synaptic complex forms prior to cleavage. Transposase made a staggered cleavage at the P-element termini that is novel for all known site-specific endonucleases: the 3' cleavage site is at the end of the P-element, whereas the 5' cleavage site is 17 bp within the P-element 31-bp inverted repeats. The P-element termini were protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable protein complex remains bound to the element termini after cleavage. These data are consistent with a cut-and-paste mechanism for P-element transposition and may explain why P elements predominantly excise imprecisely in vivo.