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Site-specific DNA transesterification catalyzed by a restriction enzyme

Lookup NU author(s): Professor Bernard Connolly

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Abstract

Most restriction endonucleases use Mg2+ to hydrolyze phosphodiester bonds at specific DNA sites. We show here that Bfil, a metal-independent restriction enzyme from the phospholipase D superfamily, catalyzes both DNA hydrolysis and transesterification reactions at its recognition site. In the presence of alcohols such as ethanol or glycerol, it attaches the alcohol covalently to the 5′ terminus of the cleaved DNA. Under certain conditions, the terminal 3′-OH of one DNA strand can attack the target phosphodiester bond in the other strand to create a DNA hairpin. Transesterification reactions on DNA with phosphorothioate linkages at the target bond proceed with retention of stereoconfiguration at the phosphorus, indicating, uniquely for a restriction enzyme, a two-step mechanism. We propose that Bfil first makes a covalent enzyme-DNA intermediate, and then it resolves it by a nucleophilic attack of water or an alcohol, to yield hydrolysis or transesterification products, respectively. © 2007 by The National Academy of Sciences of the USA.

Publication metadata

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

Publication type: Article

Publication status: Published

Journal: Proceedings of the National Academy of Sciences of the United States of America

Year: 2007

Volume: 104

Issue: 7

Pages: 2115-2120

Print publication date: 13/02/2007

ISSN (print): 0027-8424

ISSN (electronic): 1091-6490

Publisher: National Academy of Sciences

URL: http://dx.doi.org/10.1073/pnas.0608689104

DOI: 10.1073/pnas.0608689104

PubMed id: 17267608

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