Polymerase-Catalysed Incorporation of Glucose Nucleotides into a DNA Duplex

The enzymatic recognition of six-membered ring nucleoside triphosphates-in particular the 6'-triphosphates of (beta-D-glucopyranosyl)thymine, (2',3'-dideoxy-beta-D-glucopyranosyl)thymine, (3'.4'-dideoxy-beta-D-glucopyranosyl)thymine and (2',3'-dideoxy-beta-D-glucopyranosyl)adenine-was investigated. Despite the facts that the pyranose nucleic acids obtained by polymerisation of these monomers do not hybridise in solution with DNA and that the geometry of it DNA strand in a natural duplex differs front that of a pyranose nucleic acid, elongation of the DNA duplex,with all four nucleotide analogues by Vent (exo(-)) polymerase was observed. Modelling experiments showed that hydrogen bonds are formed when 2',3'-dideoxy-beta-homo-T building blocks or beta-D-gluco-T building blocks are incorporated opposite adenosine residues in the template but not when they are incorporated opposite thymine residues in the template. The model shows a near perfect alignment of a secondary hydroxy group at the end of the primer and the alpha-phosphate group of the incoming triphosphate. The results of these experiments provide new information on the role of the active site of the enzyme in the polymerisation reaction.