FokI requires two specific DNA sites for cleavage
FokI requires two specific DNA sites for cleavage
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DOI：
10.1006/jmbi.2001.4635
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发表时间：
2001-05-25
影响因子：
5.6
通讯作者：
Aggarwal, AK
中科院分区：
文献类型：
--
作者：
Vanamee, ÉS;Santagata, S;Aggarwal, AK
FokI是一种二段限制性内切酶，它识别非回文DNA序列，然后在该序列外进行双链切割，留下5‘端的悬垂。早期的动力学和结晶学研究表明，FokI可能是一种二聚体。在这里，我们使用动态光散射、凝胶过滤和分析超速离心法证明，FOKI只有在二价金属离子存在的情况下才会发生二聚化。此外，对DNA结合复合体的分析表明，识别序列的两个拷贝被结合到二聚体复合体中，该复合体的形成对于充分激活切割是必不可少的。这些结果对单体II型核酸内切酶实现高保真的机制具有广泛的意义。(C)2001年学术出版社。
FokI is a bipartite restriction endonuclease that recognizes a non-palindromic DNA sequence, and then makes double-stranded cuts outside of that sequence to leave a 5' overhang. Earlier kinetic and crystallographic studies suggested that FokI might function as a dimer. Here, we show, using dynamic light-scattering, gel-filtration and analytical ultracentrifugation, that FokI dimerizes only in the presence of divalent metal ions. Furthermore, analysis of the DNA-bound complex reveals that two copies of the recognition sequence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have broad implications for the mechanism by which monomeric type II endonucleases achieve high fidelity. (C) 2001 Academic Press.