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Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme

Lookup NU author(s): Professor Bernard Connolly

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Abstract

Restriction endonucleases catalyse DNA cleavage at specific sites. The BfiI endonuclease cuts DNA to give staggered ends with 1-nt 3'-extensions. We show here that BfiI can also fill in the staggered ends: while cleaving DNA, it can add a 2'-deoxynucleoside to the reaction product to yield directly a blunt-ended DNA. We propose that nucleoside incorporation proceeds through a two-step reaction, in which BfiI first cleaves the DNA to make a covalent enzyme-DNA intermediate and then resolves it by a nucleophilic attack of the 3'-hydroxyl group of the incoming nucleoside, to yield a transesterification product. We demonstrate that base pairing of the incoming nucleoside with the protruding DNA end serves as a template for the incorporation and governs the yield of the elongated product. The efficiency of the template-directed process has been exploited by using BfiI for the site-specific modification of DNA 5'-termini with an amino group using a 5'-amino-5'-deoxythymidine.

Publication metadata

Author(s): Sasnauskas G, Connolly BA, Halford SE, Siksnys V

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2008

Volume: 36

Issue: 12

Pages: 3969-3977

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: http://dx.doi.org/10.1093/nar/gkn343

DOI: 10.1093/nar/gkn343

Notes: Journal article

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