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Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation

Li, Y; Mitaxov, V; Waksman, G; (1999) Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. P NATL ACAD SCI USA , 96 (17) 9491 - 9496.

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Abstract

The Tag DNA polymerase is the most commonly used enzyme in DNA sequencing. However, all versions of Tag polymerase are deficient in two respects: (i) these enzymes incorporate each of the four dideoxynucleoside 5' triphosphates (ddNTPs) at widely different rates during sequencing (ddGTP, for example, is incorporated 10 times faster than the other three ddNTPs), and (ii) these enzymes show uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles, respectively. We have determined the crystal structures of all four ddNTP-trapped closed ternary complexes of the large fragment of the Tag DNA polymerase (Klentaq1). The ddGTP-trapped complex structure differs from the other three ternary complex structures by a large shift in the position of the side chain of residue 660 in the O helix, resulting in additional hydrogen bonds being formed between the guanidinium group of this residue and the base of ddGTP, When Arg-660 is mutated to Asp, Ser, Phe, Tyr, or Leu, the enzyme has a marked and selective reduction in ddGTP incorporation rate, As a result, the G track generated during DNA sequencing by these Tag polymerase variants does not terminate prematurely, and higher molecular-mass G bands are detected, Another property of these Tag polymerase variants is that the sequencing patterns produced by these enzymes are remarkably even in band-intensity and peak-height distribution, thus resulting in a significant improvement in the accuracy of DNA sequencing.

Type: Article
Title: Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation
Keywords: x-ray crystallography, DNA sequencing, TERMINATORS, BIAS
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/125614
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