MnlI - The member of H-N-H subtype of Type IIS restriction endonucleases
MnlI - The member of H-N-H subtype of Type IIS restriction endonucleases
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DOI：
10.1016/j.bbapap.2005.06.006
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发表时间：
2005-08-10
影响因子：
3.2
通讯作者：
Lubys, A
中科院分区：
文献类型：
--
作者：
Kriukiene, E;Lubiene, J;Lubys, A
IIS型限制性内切核酸酶穆尔识别非回文核苷酸序列5 '-CCTC（N）7/6向下箭头，并切割DNA链，如箭头所示。对编码穆尔酶切修饰系统的基因进行克隆和序列测定。它包括N6-甲基腺嘌呤和C5-甲基胞嘧啶甲基转移酶和限制性内切核酸酶。生物化学研究表明，穆尔限制性内切酶切割双链和单链DNA，它更喜欢不同的金属离子水解这些底物。研究表明，Mg 2+离子是特异性切割双链DNA所需的，而Ni 2+和一些其他过渡金属离子是特异性切割单链DNA所需的。穆尔限制性内切酶的C-末端部分显示与细菌毒素Colicin E7和Colicin E9的H-N-H型溶核结构域的有趣的相似性。保守序列基序306 Rx（3）ExHHx（14）Nx（8）H中丙氨酸的替换大大降低了穆尔的比活性，有些突变甚至使该酶完全失活。然而，这些突变都不影响MnII与特定DNA的结合，也不影响其寡聚状态。我们解释了所提出的实验证据作为一个建议，基序306 Rx（3）ExHHx（14）Nx（8）H代表的活性位点的穆尔。因此，MnII似乎是具有H-N-H型活性位点的IIS型成员。(c)2005 Elsevier B. V.保留所有权利。
The Type IIS restriction endonuclease Mull recognizes the non-palindromic nucleotide sequence 5'-CCTC(N)7/6 down arrow and cleaves DNA strands as indicated by the arrow. The genes encoding Mull restriction-modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that Mull restriction endonuclease cleaves double- and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of Mull restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx(3)ExHHx(14)Nx(8)H greatly reduced specific activity of Mull, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnII binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx(3)ExHHx(14)Nx(8)H represents the active site of Mull. Consequentially, MnII seems to be the member of Type IIS with the active site of the H-N-H type. (c) 2005 Elsevier B.V. All rights reserved.