Figure 1.
Overview of PolC structure and domain organization.
A. Structure of Gka-PolC-ΔNΔExo shown (PDB ID: 3F2B). Domains are color coded as in Figure 1B. OB domain is not shown for simplicity. The linker connecting the N-palm and PHP domains is shown (yellow). B. Sequence alignment showing domain organization of C-family polymerases.
Figure 2.
Minimal single-nucleotide incorporation reaction pathway for DNA polymerases.
Abbreviations used were: E, DNA polymerase; D0, unextended DNA; D1, DNA extended by one base-pair; PPi, inorganic pyrophosphate. Dashed arrow indicates the polymerase entering another round of catalysis.
Figure 3.
A 10% SDS-polyacrylamide gel stained with Coomassie R-250 showing purified Sau-PolC-ΔNΔExo obtained after size exclusion chromatography. (a) Kaleidoscope pre-stained marker. (b) 2.5 µM purified Sau-PolC-ΔNΔExo.
Figure 4.
Primer extension assays for optimizing enzymatic activity of Sau-PolC-ΔNΔExo.
(A) Duplex DNA sequence used for all primer extension assays performed in this study. “*/” at 5′ end of primer indicates 6-FAM label. (B) Effect of pH (C) Effect of NaCl concentration (D) Effect of Mg2+ concentration and (E) Effect of temperature on Sau-PolC-ΔNΔExo activity. Primer extension assays were carried out under steady-state conditions by adding 1 mM dTTP (the correct incoming dNTP) to a pre-incubated solution of 400 nM p/t DNA and 1 nM Sau-PolC-ΔNΔExo. Reactions were quenched after 2 minutes by addition of an equal volume of 250 mM EDTA. Unextended and extended primers were separated by gel electrophoresis on a 17% denaturing TBE-acrylamide gel. Fraction of primer DNA extended was determined by measuring the relative intensity of the extended primer band with respect to the total labeled DNA (extended and unextended primer).
Figure 5.
Steady-state kinetic analysis of Sau-PolC-ΔNΔExo.
Primer extension assays were performed by adding dTTP (final concentration range 9.38 to 300 µM) to a final concentrations of 2.5 µM p/t DNA and 16.5 nM active Sau-PolC-ΔNΔExo. The reactions were quenched at different time intervals with 250 mM EDTA. (A) A typical time course of primer extension followed during the steady-state kinetic assays (final [dTTP] was 300 µM). The concentration of primer extended was plotted against time and fit to the steady-state equation (Equation 1). (B) Michaelis-Menten plot for Sau-PolC-ΔNΔExo. The observed rates of primer extension were plotted as a function of the dTTP concentration. The resulting plot was fit to the Michaelis-Menten equation (Equation 2). From the fit, steady-state rate constant (kcat) was calculated to be 17±1 s−1 and Michaelis constant for dNTP (KMdNTP) was determined to be 43±7 µM.
Figure 6.
Determination of the DNA dissociation rate from polymerase ⋅ DNA binary complex (koff).
(A) Schematic representation of the experimental procedure. (B) Plot of product formed vs time. The data were fit to a single exponential equation (Equation 3). The rate of decrease in product formation (which is equivalent to the rate of dissociation of p/t DNA from Sau-PolC-ΔNΔExo ⋅ p/t DNA binary complex (koff)) was 150±30 s−1.
Figure 7.
Pre-steady-state kinetics and active site titration of Sau-PolC- ΔNΔExo.
(A) A time course of primer extension under pre-steady-state condition in the presence (⋅) and absence () of unlabelled p/t DNA acting as an enzyme trap. 35 µM dTTP (with or without 48 µM of unlabelled p/t DNA) was added to 150 nM Sau-PolC-ΔNΔExo (corresponding to an active Sau-PolC-ΔNΔExo concentration of 100 nM) and 80 nM p/t DNA (all concentrations are final). In the absence of the trap, the time course was biphasic in nature and the data were fit to the full burst equation (Equation 4). The rate of the fast phase was 150±30 s−1 and that of the slower phase was 8.5±1 s−1, [ED]A was found to be 12±1 nM. In the presence of the trap, the time course was monophasic and the data were fit to a single exponential equation, with a rate of 300±14 s−1 and an amplitude of 11.5±0.5 nM. The data can also be fit equally well to the full burst equation, but the data were not sufficient to justify using the more complex model. (B) A representative set of primer extension assays performed during active site titration. Time resolved primer extension assays were performed using 150 nM Sau-PolC-ΔNΔExo, 1 mM dTTP and varying concentrations of p/t DNA (⋅ 40 nM, ▪ 80 nM,
160.1 nM,♦ 284.76 nM,<$>\raster="rg1"<$> 379.69 nM, ○ 506.25 nM, +675 nM and ×900 nM). The concentration of extended primer was plotted versus time and data were fit to the full burst equation (Equation 4). For ease of understanding, the background primer extension has been deducted from each time course. (C) A plot of the concentrations of pre-formed active enzyme-DNA complex getting converted to product before turnover ([ED]A) versus DNA concentration was fit to a quadratic equation (Equation 5). KDDNA was determined to be 390±70 nM and the concentration of active Sau-PolC-ΔNΔExo was found to be 100±8 nM.
Table 1.
Comparison of kinetic parameters of different polymerases.
Figure 8.
Determination of KDdNTP of Sau-PolC- ΔNΔExo.
(A) A representative set of primer extension assays performed during KDdNTP determination for Sau-PolC-ΔNΔExo. The reactions were performed with 804 nM active Sau-PolC-ΔNΔExo, 50 nM p/t DNA and various concentrations of dTTP (◊ 1.17 µM, ○ 4.69 µM, ♦ 9.4 µM, ▾ 18.75 µM, ▴ 30.14 µM, ▪ 50 µM and • 75 µM). The concentrations of extended primers were plotted against time and the plots were fit to the full burst equation (Equation 4). (B) A plot of the observed rates of the fast phase (k1) versus [dTTP]. The data were fit to a hyperbolic equation (Equation 6). From the fit, KDdNTP was determined to be 3.2±0.9 µM and maximum rate of the burst (kpol) was found to be 178±9 s−1. R2 value for this fit was 0.75. (C) An enlarged view of panel (B) of up to 15 µM of [dTTP]. (D) A plot of [ED]A versus [dTTP] was fit to a hyperbolic equation (Equation 7). From the fit, KDdNTP was found to be 4.0±0.3 µM and the maximum [ED]A was 36±0.6 nM. R2 value for this fit was 0.98.
Figure 9.
Simulation of kinetic pathway of Sau-PolC- ΔNΔExo.
(A) The three-step reaction mechanism used for the simulation. Values obtained for the different rate constants are shown alongside the appropriate step. Rate of dNTP association to the E⋅D0 binary complex was assumed to be diffusion limited and accordingly the second order rate constant for this step was fixed at 100 µM−1s−1. (B) Simulated curves generated for the representative dataset shown in Figure 8A superimposed on the raw data (concentrations of dTTP shown are ○ 1.17 µM, ♦ 4.69 µM, • 9.4 µM, ▽ 18.75 µM, ▴ 30.14 µM, ▪ 50 µM and ×75 µM). (C) 3-D confidence contours for the various rate constants determined from the simulation. For each case the search was carried out up to a sum of squares error (SSE) that is 2-fold higher than the minimum SSE. The upper and lower limits of each parameter were determined using an SSE threshold of 1.2.
Figure 10.
Minimal enzymatic pathway for Sau-PolC- ΔNΔExo.
The kinetic parameters determined are shown alongside the corresponding steps of the pathway. KDdNTP, forward and reverse rates of chemistry and rate of PPi release were derived from simulation of the reaction pathway. The rate of enzyme-DNA association (kon) was calculated from the KDDNA and koff using the relation that KDDNA = koff/kon. It should be noted that although nucleotide binding and PPi release are each shown as single steps, they may in fact be comprised of more than one elementary step, such as a conformational change in the polymerase accompanying substrate binding and product release.