A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
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DOI：
10.1093/nar/gki921
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Kumar, S
中科院分区：
文献类型：
--
作者：
Guan, C;Kumar, S
通过反式剪接内含肽系统产生含有T7内切核酸酶I的单个正确折叠催化结构域（SCD）的稳定异源二聚体蛋白。正如先前提出的模型所预测的，纯化的SCD蛋白对正常线性DNA起非特异性切口核酸内切酶的作用。SCD保留了识别和切割切口或链交叉位点附近的偏离DNA双螺旋的能力。因此，我们推断对于天然T7内切核酸酶I观察到的非特异性和切口位点切割活性（与拆分活性不同）是由于催化结构域的不协调作用。T7 Endo I的带正电荷的C-末端对于SCD的酶活性是必需的，因为它对于天然酶是必需的。我们建议，优先的天然酶的决议反应是通过协同性在其两个催化结构域的结合时，提出了两个跨四向交界处或十字形结构的武器。
A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure.