How is a selective enzyme made even more selective? With a combinatorial approach to redesign an enzyme motif (see figure; red=O, blue=N), the mismatch extension selectivity of DNA polymerases was increased. The results impart new and deeper insight into the mechanisms of DNA polymerases and provide new tools for applications such as efficient genotyping.
DNA polymerase fidelity is of immense biological importance due to the fundamental requirement for accurate DNA synthesis in both replicative and repair processes. Subtle hydrogen-bonding networks between DNA polymerases and their primer/template substrates are believed to have impact on DNA polymerase selectivity. We show that deleting defined interactions of that kind by rationally designed hydrophobic substitution mutations can result in a more selective enzyme. Furthermore, a single-atom replacement within the DNA substrate through chemical modification, which leads to an altered acceptor potential and steric demand of the DNA substrate, further increased the selectivity of the developed systems. Accordingly, this study about the impact of hydrophobic alterations on DNA polymerase selectivity--enzyme and substrate wise--further highlights the relevance of shape complementary and polar interactions on DNA polymerase selectivity.
Wie macht man ein selektives Enzym noch selektiver? Durch Modifizierung eines Enzymmotivs (siehe Abbildung; rot O, blau N) mithilfe eines kombinatorischen Ansatzes ließ sich die Selektivität der Fehlpaarungsverlängerung von DNA‐Polymerasen verbessern. Die Ergebnisse ermöglichen tiefere Einsichten in die Mechanismen von DNA‐Polymerasen und stellen neue Werkzeuge, z. B. für eine effiziente Genotypisierung, zur Verfügung.
The development of new synthetic probes to study the mechanism of Uracil-DNA glycosylase is described. 4¢-C-Alkylated 2¢-deoxyuridines were synthesized and incorporated site-specifically into oligonucleotides. These compounds were subsequently employed in functional studies of the enzyme and proved to be functional competent.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.