Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.

Biochemistry (1991), Volume 30, Page 511

Abstract:

The elementary steps of DNA polymerization catalyzed by T7 DNA polymerase have been resolved by transient-state analysis of single nucleotide incorporation, leading to the complete pathway: [formula: see text] where E, D, N, and P represent T7 DNA polymerase, DNA primer/template, deoxynucleoside triphosphate, and inorganic pyrophosphate, respectively. A DNA primer/template consisting of a synthetic 25/36-mer has been used as a substrate for correct nucleotide incorporation of dTTP in all the experiments. The rate constants and equilibrium constants of each step have been established by direct measurement of individual reactions and fit by computer simulation of the data to obtain a single set of rate constants accounting for all the data. Analysis of the single-turnover kinetics provided measurements of equilibrium dissociation constants for 25/36-mer, dTTP, and PPi equal to 18 nM (koff/kon), 18 microM (k-1/k1), and 2 mM (k5/k-5), respectively. The rate-limiting step during single-nucleotide incorporation has been identified as a conformational change, E.Dn.N----E'.Dn.N, which occurs at a rate of 300 s-1 (k2) upon binding of the correct dNTP. Accordingly, tighter binding of the transition states for the reaction resulting from the conformational change facilitates the phosphodiester bond formation. The chemical step itself was excluded as the rate-limiting step because of the small phosphothioate elemental effect. An observed rate constant of 70 s-1 for dTTP (alpha S) incorporation suggest that the chemical step (k3) occurs at a fast rate, greater than or equal to 9000 s-1. Following chemistry, the resulting ternary complex, E'.Dn+1.P, undergoes a second conformational change at a rate of 1200 s-1 (k4), leading to release of PPi and translocation of the DNA to continue subsequent cycles of polymerization. The rate constants of the reverse steps, 100 s-1 (k-2), greater than or equal to 18,000 s-1 (k-3) and 18 s-1 (k-4), were derived as fits to the data based upon simulation of single-turnover kinetics of pyrophosphorolysis including measurements of pyrophosphate exchange and the overall equilibrium constant of 1.0 x 10(4) for elongation of E.25/36-mer and analysis of the kinetics of the pulse-chase experiment. These studies provide the first complete and self-consistent thermodynamic descriptions of DNA polymerase and establish the basis for quantitative assessment of the reactions contributing to its extraordinary fidelity.(ABSTRACT TRUNCATED AT 400 WORDS)

Polymerases:

Topics:

Kinetic Parameters, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 (exo-) Patel SS1991 3-5' Exonuclease (proofreading) No
T7 (exo-) Patel SS1991 Cloned or native Cloned in E. coli
T7 (exo-) Patel SS1991 KM 800nM Reaction: 3-5' Exonuclease; Substrate: DNA template
T7 (exo-) Patel SS1991 Vmax 1 /second Reaction: Pyrophosphorolysis
T7 (exo-) Patel SS1991 Vmax 242 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
T7 (exo-) Patel SS1991 Vmax 0.00069 /second Reaction: 3-5' Exonuclease
T7 (exo-) Patel SS1991 Kd 21uM Reaction: Nucleotide incorporation; Substrate: dNTPs
T7 (exo-) Patel SS1991 Kd 16nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
T7 (exo-) Patel SS1991 Kd 2000uM Reaction: Pyrophosphorolysis
T7 Patel SS1991 3-5' Exonuclease (proofreading) Yes
T7 Patel SS1991 Cloned or native Cloned in E. coli
T7 Patel SS1991 Full length or truncated Full length
T7 Patel SS1991 Processivity 1500bp
T7 Patel SS1991 KM 690nM Reaction: 3-5' Exonuclease; Substrate: DNA template
T7 Patel SS1991 Vmax 287 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
T7 Patel SS1991 Vmax 105 /second Reaction: 3-5' Exonuclease
T7 Patel SS1991 Kd 23nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
T7 Patel SS1991 Kd 18uM Reaction: Nucleotide incorporation; Substrate: dNTPs
T7 Patel SS1991 Kd 2000uM Reaction: Pyrophosphorolysis

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