Kinetics of error generation in homologous B-family DNA polymerases

Abstract

The kinetics of forming a proper Watson-Crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the B Family replicative DNA polymerase from bacteriophage T4. Structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage RB69. In this work, the homologous enzymes from RB69 and T4 were compared in parallel reactions to determine the relative abilities of the two polymerases to incorporate correct nucleotides as well as to form improper pairings. The kinetic rates for three different exonuclease mutants for each enzyme were measured for incorporation of an A opposite T and an A opposite furan as well as for the formation of A:C and T:T mismatches. The T4 exonuclease mutants were all approximately 2- to 7-fold more efficient than the corresponding RB69 exonuclease mutants depending on whether a T or furan was in the templating position and which exonuclease mutant was used. The rates for mismatch formation by T4 were significantly reduced compared with incorporation opposite furan, much more so than the corresponding RB69 mutant. These results show that there are kinetic differences between the two enzymes but they are not large enough to preclude structural assumptions for T4 DNA polymerase based on the known structure of the RB69 DNA polymerase.

Figure 1

Exonuclease active site of T4 DNA polymerase (21) (PDB: 1NOY). The structure of the exonuclease active site is shown with the four conserved catalytic residues. The two divalent metal ions are shown as spheres. Inter-atomic distances <3.5 Å are shown as red, dashed lines and those >3.5 Å are shown in blue. This figure was made with PyMOL (37).

Figure 2

Kinetic analysis of dAMP incorporation opposite furan (F). (A) Representative gels and progress curves are shown for the fastest mutants at the highest concentration of dATP used in the experiments (4 mM dATP) plotted against reaction time. These curves were best fit to a single exponential equation [$P=A*(1-{\text{e}}^{-k_1*t})+C$]. (B) Calculation of polymerase rate constants and dissociation constants for the incoming nucleotide. The observed rate constants (k_obs) for incorporation of A opposite F were plotted against dATP concentrations and fit to a hyperbola to give values for k_pol and K_{D dATP} for all three exonuclease mutants of T4 and RB69 DNA polymerases. (closed triangle = T4 D219A, open triangle = RB69 D222A, closed circle = T4 D112A/E114A, open circle = RB69 D114A/E116A, closed square = T4 D324A, open square = RB69 D327A).

Figure 3

Kinetic analysis of dAMP incorporation opposite a templating T. (A) Representative gels and progress curves are shown for the fastest mutants at the highest concentration of dATP used in the experiments (0.4 mM dATP) plotted against reaction time. These curves were best fit to a double exponential equation [$P=A*(1-{\text{e}}^{-k_1*t})+B*(1-{\text{e}}^{-k_2*t})+C$]. The inset shows the progress curve for the slowest T4 mutant (D324A) and a comparison between a double exponential curve fit (solid line) and a single exponential curve fit (dashed line). (B) Calculation of polymerase rate constants and dissociation constants for the incoming nucleotide. The observed, fast rate constants (k_obs) for incorporation of A opposite T were plotted against dATP concentrations and fit to a hyperbola to give values for k_pol and K_{D dATP} for all three exonuclease mutants of T4 and RB69 DNA polymerases. (Closed triangle = T4 D219A, open triangle = RB69 D222A, closed circle = T4 D112A/E114A, open circle = RB69 D114A/E116A, closed square = T4 D324A, open square = RB69 D327A.).

Figure 4

Kinetic analysis of mismatch formation. The progress curves for incorporating an A opposite C or T opposite T are shown as the percentage of primer extension plotted against reaction times. Both curves were fit to a single exponential equation [$P=A*(1-{\text{e}}^{-k_1*t})+C$]. (Closed triangle = T4 D219A, open triangle = RB69 D222A.).