Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme.
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.
Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania.
Giedrius
G
Sasnauskas
Bernard A
BA
Connolly
Stephen E
SE
Halford
Virginijus
V
Siksnys
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D003853:Deoxyribonucleosides / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013698:Templates, Genetic
D003853:Deoxyribonucleosides; D004247:DNA; C113635:endodeoxyribonuclease BfiI; D015252:Deoxyribonucleases, Type II Site-Specific