Y. Li, V. Mitaxov, and G. Waksman.
Structure-based design of novel Taq DNA polymerases with improved properties
of dideoxynucleotide incorporation
The Taq DNA polymerase is the most commonly used enzyme in DNA
sequencing. However, all versions of Taq polymerase are deficient in two
respects: 1) these enzymes incorporate each of the four dideoxynucleoside 5¹
triphosphates (ddNTPs) at widely different rates during sequencing (ddGTP for
example is incorporated ten-fold faster than the other three ddNTPs) and 2) 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 Taq 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 R660 in the O helix, resulting
in additional hydrogen bonds being formed between the guanidinium group of
this residue and the base of ddGTP. When residue 660 is mutated to Asp, Ser, Phe,
Tyr, or Leu, the enzyme demonstrates a marked and selective reduction in
ddGTP incorporation rate. As a result, the G track generated during DNA
sequencing by these Taq polymerase variants does not terminate prematurely
and higher molecular weight G bands are detected. Another property of these
Taq 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.
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