Nucleic Acids Research Advance Access published online on May 30, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn343
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Nucleic Acid Enzymes |
Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme
1Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania, 2Institute for Cell and Molecular Biosciences (ICaMB) University of Newcastle, Newcastle upon Tyne NE2 4HH and 3The DNA-Proteins Interaction Unit, Department of Biochemistry, School Of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
*To whom correspondence should be addressed: Tel: +370 5 2602108; Fax: +370 5 2602116; Email: siksnys{at}ibt.lt
Received April 18, 2008. Revised May 9, 2008. Accepted May 12, 2008.
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.