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

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' triphosphat es (ddNTPs) at widely different rates during sequencing (ddGTP, for example , is incorporated 10 times faster than the other three ddNTPs), and (ii) th ese enzymes show uneven band-intensity or peak-height patterns in radio-lab eled 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-trapp ed complex structure differs from the other three ternary complex structure s 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 gu anidinium group of this residue and the base of ddGTP, When Arg-660 is muta ted to Asp, Ser, Phe, Tyr, or Leu, the enzyme has a marked and selective re duction in ddGTP incorporation rate, As a result, the G track generated dur ing DNA sequencing by these Tag polymerase variants does not terminate prem aturely, and higher molecular-mass G bands are detected, Another property o f these Tag polymerase variants is that the sequencing patterns produced by these enzymes are remarkably even in band-intensity and peak-height distri bution, thus resulting in a significant improvement in the accuracy of DNA sequencing.