Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity
Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity
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DOI：
10.1016/j.jmb.2008.07.010
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发表时间：
2008-09-26
影响因子：
5.6
通讯作者：
Gimble, Frederick S.
中科院分区：
文献类型：
--
作者：
Niu, Yan;Tenney, Kristen;Gimble, Frederick S.
目前可用于实验室程序和体内应用的链特异性缺口核酸内切酶的数量有限，而且没有一种酶对复杂基因组中的缺口单一靶点具有足够的特异性。归巢核酸内切酶具有极高的靶标特异性，使其成为工程高特异性缺口核酸内切酶的候选分子。I-SCEI是一种单体定位酶，它识别一个18bp的不对称靶序列，并切割两条DNA链，留下4个核苷酸的3‘-突出端。在以质粒为底物的单周转实验中，I-SCEI在超螺旋DNA转化为线状DNA的过程中产生了瞬时的开环中间体，表明该酶是依次切割这两条DNA链的。一种新的发夹底物被用来证明尽管野生型I-SCEI首先切割顶端或底端的DNA链以产生两个缺口的DNA中间体，但该酶具有切割底链的偏好。动力学数据符合平行顺序反应机理。两个假对称残基Lys122和Lys223的取代分别显著减少了顶链和底链的切割，从而产生了具有显著的链和序列特异性的切割活性的酶。这两个活性部位是部分相互依赖的，因为一个部位的改变会影响另一个部位。动力学分析与I-SCEI/DNA络合物的X射线晶体结构一致，揭示了赖氨酸在建立重要的溶剂网络中的作用，其中包括被认为攻击剪刀式磷酸二酯键的亲核水分子。(C)2008爱思唯尔有限公司。保留所有权利。
The number of strand-specific nicking endonucleases that are currently available for laboratory procedures and applications in vivo is limited, and none is sufficiently specific to nick single target sites within complex genomes. The extreme target specificity of homing endonucleases makes them attractive candidates for engineering high-specificity nicking endonucleases. I-SceI is a monomeric homing enzyme that recognizes an 18 bp asymmetric target sequence, and cleaves both DNA strands to leave 3 '-overhangs of 4 bp. In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme cleaves the two DNA strands sequentially. A novel hairpin substrate was used to demonstrate that although wild-type I-SceI cleaves either the top or bottom DNA strand first to generate two nicked DNA intermediates, the enzyme has a preference for cleaving the bottom strand. The kinetics data are consistent with a parallel sequential reaction mechanism. Substitution of two pseudo-symmetric residues, Lys122 and Lys223, markedly reduces top and bottom-strand cleavage, respectively, to generate enzymes with significant strand- and sequence-specific nicking activity. The two active sites are partially interdependent, since alterations to one site affect the second. The kinetics analysis is consistent with X-ray crystal structures of I-SceI/DNA complexes that reveal a role for the lysines in establishing important solvent networks that include nucleophilic water molecules thought to attack the scissile phosphodiester bonds. (C) 2008 Elsevier Ltd. All rights reserved.