Taq DNA Polymerase Mutants and 2'-Modified Sugar Recognition

Biochemistry
Hayley J SchultzAaron M Leconte

Abstract

Chemical modifications to DNA, such as 2' modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2'-modified DNA nucleotides. While these mutant enzymes recognize these modified nucleotides, they are not capable of synthesizing full length modified DNA; thus, further engineering is required for these enzymes. Here, we describe comparative biochemical studies that identify useful, but previously uncharacterized, properties of these enzymes; one enzyme, SFM19, is able to recognize a range of 2'-modified nucleotides much wider than that previously examined, including fluoro, azido, and amino modifications. To understand the molecular origins of these differences, we also identify specific amino acids and combinations of amino acids that contribute most to the previously evolved unnatural activity. Our data suggest that a negatively charged amino acid at 614 and mutation of the steric gate residue, E615, to glycine make up the optimal combination for modified oligonucleotide sy...Continue Reading

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Citations

May 21, 2016·Frontiers in Chemistry·María A DellafioreAdolfo M Iribarren
Feb 6, 2017·Chembiochem : a European Journal of Chemical Biology·Sydney L RosenblumAaron M Leconte
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Apr 7, 2021·Chembiochem : a European Journal of Chemical Biology·Sana OhashiHiroshi Abe
May 1, 2021·RSC Chemical Biology·Adeline EspinasseErin E Carlson

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