Higgins L S, Besnier C, Kong H
New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA.
Nucleic Acids Res. 2001 Jun 15;29(12):2492-501. doi: 10.1093/nar/29.12.2492.
N.BstNBI is a nicking endonuclease that recognizes the sequence GAGTC and nicks the top strand preferentially. The Type IIs restriction endonucleases PleI and MlyI also recognize GAGTC, but cleave both DNA strands. Cloning and sequencing the genes encoding each of these three endonucleases discloses significant sequence similarities. Mutagenesis studies reveal a conserved set of catalytic residues among the three endonucleases, suggesting that they are closely related to each other. Furthermore, PleI and MlyI contain a single active site for DNA cleavage. The results from cleavage assays show that the reactions catalyzed by PleI and MlyI are sequential two step processes. The double-stranded DNA is first nicked on one DNA strand and then further cleaved on the second strand to form linear DNA. Gel filtration analysis shows that MlyI dimerizes in the presence of a cognate DNA and Ca(2+) whereas N.BstNBI remains a monomer, implicating dimerization as a requisite for the second strand cleavage. We suggest that N.BstNBI, MlyI and PleI diverged from a common ancestor and propose that N.BstNBI differs from MlyI and PleI in having an extremely limited second strand cleavage activity, resulting in a site-specific nicking endonuclease.
N.BstNBI是一种切口核酸内切酶，它识别序列GAGTC并优先切割上链。IIs型限制性内切酶PleI和MlyI也识别GAGTC，但切割两条DNA链。对编码这三种内切酶的基因进行克隆和测序，发现它们具有显著的序列相似性。诱变研究揭示了这三种内切酶中一组保守的催化残基，表明它们彼此密切相关。此外，PleI和MlyI含有一个用于DNA切割的单一活性位点。切割分析结果表明，PleI和MlyI催化的反应是连续的两步过程。双链DNA首先在一条DNA链上产生切口，然后在第二条链上进一步切割形成线性DNA。凝胶过滤分析表明，MlyI在同源DNA和Ca(2+)存在下会二聚化，而N.BstNBI仍然是单体，这意味着二聚化是第二条链切割的必要条件。我们认为N.BstNBI、MlyI和PleI起源于一个共同的祖先，并提出N.BstNBI与MlyI和PleI的不同之处在于其第二条链切割活性极其有限，从而形成了一种位点特异性切口核酸内切酶。
