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Lookup NU author(s): Professor Bernard Connolly
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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.
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
Print publication date: 13/02/2007
ISSN (print): 0027-8424
ISSN (electronic): 1091-6490
Publisher: National Academy of Sciences
PubMed id: 17267608
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