SLOW RATE OF PHOSPHODIESTER BOND FORMATION ACCOUNTS FOR THE STRONG BIAS THAT TAQ DNA-POLYMERASE SHOWS AGAINST 2',3'-DIDEOXYNUCLEOTIDE TERMINATORS

Tag and T7 DNA polymerases have become basic molecular biology ''tools '' for DNA sequence analysis, However, Tag, unlike T7 DNA polymerase, is strongly biased against the incorporation of 2',3'-dideoxynucleotid e triphosphates (ddNTPs) indicating very different substrate selectivi ties. Equilibrium binding and rate constants were measured for 2',3'-d dNTPs as well as for several other 3'-substituted terminators and comp ared to 2'-deoxynucleotide substrates (dNTPs). In steady-state experim ents, Tag Pol I was strongly biased in favor of dATP(1) over ddATP inc orporation by about 700 to 1, in contrast to T7 DNA polymerase which s howed a preference of only about 4 to 1. Manganese reduced but did not eliminate selectivity against 2',3'-ddNTPs. Transient kinetic traces indicated different rate-limiting steps for substrate and terminator i ncorporation. Further mechanistic studies showed that the binding cons tants for substrates and terminators were equivalent. However, the rat e constants for phosphodiester bond formation for 2',3'-ddNTPs were 20 0-3000-fold lower than for dNTPs, Alternative terminators showed only slight improvements. The data were consistent with a model in which bo th substrates and terminators undergo ground-state binding followed by formation of a tight-binding Enz . DNA . Nucleotide complex. Immediat ely after complex formation, substrates undergo a rapid nucleoside pho sphoryl transfer reaction. However, the reaction rates for terminators were slower presumably due to misalignment of reactive groups in the active site. Thus, the strong bias that Taq DNA polymerase shows again st terminators is due to a very slow ''chemistry'' step, Such a strong bias has several kinetic consequences for DNA sequence patterns. Thes e consequences are discussed in the text.