Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity
Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity
复制标题
DOI：
10.1016/j.str.2016.03.024
复制
发表时间：
2016-06-07
期刊：
影响因子：
5.7
通讯作者：
Stoddard, Barry L.
中科院分区：
文献类型：
--
作者：
Lambert, Abigail R.;Hallinan, Jazmine P.;Stoddard, Barry L.
LAGLIDADG大范围核酸酶是用于基因组工程的DNA切割酶。虽然它们的切割特异性可以使用几种蛋白质工程和选择策略来改变，但它们的总体靶向性受到其识别位点内的中心四个碱基对的高度特异性间接识别的限制。为了研究间接序列识别的物理基础，并扩大可用于基因组工程的此类核酸酶的数量，我们确定了9种相关酶的靶位点、DNA结合结构和中心4个切割能力。随后的大范围核酸酶结合到两个不可切割的靶位点，每个含有一个单一的失活碱基对取代在其中心，晶体学分析表明，突变的碱基对的局部滑动导致DNA骨架构象的小变化，导致在一个结合位点的金属占有率的损失，消除切割活性。
LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.