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The nicking endonuclease N.BstNBI is closely related to type IIs restriction endonucleases MlyI and PleI.

ABSTRACT: 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.

SUBMITTER: Higgins LS

PROVIDER: S-EPMC55753 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

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The nicking endonuclease N.BstNBI is closely related to type IIs restriction endonucleases MlyI and PleI.

Higgins L S LS Besnier C C Kong H H

Nucleic acids research 20010601 12

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, ...[more]

PMID: 11410656

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Project description:More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.

Dataset Information

The nicking endonuclease N.BstNBI is closely related to type IIs restriction endonucleases MlyI and PleI.

Publications

The nicking endonuclease N.BstNBI is closely related to type IIs restriction endonucleases MlyI and PleI.

Similar Datasets

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