Kinetic analysis of correct nucleotide insertion by a Y-family DNA polymerase reveals conformational changes both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence

Abstract

The Sulfolobus solfataricus Y-family DNA polymerase Dpo4 is a model for translesion replication and has been used in the analysis of individual steps involved in catalysis. The role of conformational changes has not been clear. Introduction of Trp residues into the Trp-devoid wild-type protein provided fluorescence probes of these events, particularly in the case of mutants T239W and N188W. With both mutants, a rapid increase in Trp fluorescence was observed only in the case of normal base pairing (G:C), was saturable with respect to dCTP concentration, and occurred in the absence of phosphodiester bond formation. A subsequent decrease in the Trp fluorescence occurred when phosphodiester bond formation was permitted, and these rates were independent of the dCTP concentration. This step is relatively slow and is attributed to a conformational relaxation step occurring after pyrophosphate release, which was measured and shown to be fast in a separate experiment. The measured rate of release of DNA from Dpo4 was rapid and is not rate-limiting. Overall, the measurements provide a kinetic scheme for Dpo4 different than generally accepted for replicative polymerases or proposed for Dpo4 and other Y-family polymerases: the initial enzyme.DNA.dNTP complex undergoes a rapid (18 s(-1)), reversible (21 s(-1)) conformational change, followed by relatively rapid phosphodiester bond formation (11 s(-1)) and then fast release of pyrophosphate, followed by a rate-limiting relaxation of the active conformation (2 s(-1)) and then rapid DNA release, yielding an overall steady-state kcat of <1 s(-1).

FIGURE 1.

Model of Trp residues mutated within a Dpo4 ternary structures. Adapted from PDB code 2ASD (14). A, Trp modeled into Dpo4 at residue 188; color scheme: Trp (green), Dpo4 (gray), DNA (yellow), and the incoming dCTP (blue). B, Trp modeled into Dpo4 at residue 239 (same color scheme as in A).

FIGURE 2.

Steady-state fluorescence spectra of substrate binding to Dpo4-Trp mutants. A, spectra of 1 μm Dpo4 T239W before (black trace) and after (blue trace) addition of 1 μm DNA_G was added, respectively. B and C, same as in A but with Dpo4 N188W (B) and Dpo4 Y12W (C), respectively. D, free Trp control. Spectra were obtained after adding 500 μm of each dNTP separately to 1 μm free Trp: dATP (red), dTTP (green), dCTP (blue), dGTP (brown), and no dNTP (black). E, same as in part D, except 1 μm free Trp was replaced by a 1 μm Dpo4 T239W·DNA_G complex.

FIGURE 3.

Fluorescence changes of Dpo4 mutants T239W and N188W observed upon binding of dNTPs and catalysis. The relevant portion of the DNA sequence within the region of dNTP binding is shown above the graphs. All dNTPs were added to a final concentration of 500 μm: dATP (red), dTTP (black), dCTP (blue), and dGTP (green). A, G as the incipient template base and C the next, +1 base. The addition of dCTP or dGTP increased the fluorescence of Dpo4 T239W in the absence of phosphodiester bond formation (3′-deoxy primer terminus). B, when phosphodiester bond formation occurred (3′-OH primer terminus), the fluorescence increase seen with Dpo4 T239W was followed by a decrease (adding dCTP opposite G). C, an experiment similar to that of C was done, but the sequence context was changed to insert dNTPs opposite C (instead of G). D, with Dpo4 N188W as the enzyme (using the same sequence context as in part A), fluorescence changes were only seen when the correct dNTP was added.

FIGURE 4.

DNA off-rates estimated by changes in Trp fluorescence of Dpo4 mutants. A, a Dpo4·DNA_G complex was rapidly mixed with a 5-fold molar excess of wild-type Dpo4 as a trap and the change in fluorescence upon release of DNA_G from T239W was observed, k_off = 73 ± 3 s^-1. B, a wild-type Dpo4·DNA_G complex (blue trace) or DNA_G alone (black trace) was rapidly mixed with a 5-fold molar excess of Dpo4 T239W as the trap, and the fluorescence increase observed upon Dpo4 T239W binding of the released DNA from wild-type Dpo4 was recorded, k_off = 70 ± 4 s^-1. C, as in B, DNA off-rates from wild-type Dpo4 binary (Dpo4·DNA_G, blue trace) and ternary (Dpo4··dCTP, red trace) complexes upon binding to Dpo4 Y12W were recorded, k_off(binary) = 99 ± 6 s^-1 and k_off(ternary) = 16 ± 2 s^-1.

FIGURE 5.

Fluorescence changes observed upon dCTP binding to a Dpo4 T239W· complex. A, stopped-flow fluorescence changes induced by addition of dCTP opposite template G: 15 μm (red); 30 μm (orange); 50 μm (green); 120 μm (blue); 240 μm (violet); and 500 μm (brown). B, rates of dCTP-induced fluorescence changes and the ground state K_d,dCTP opposite template G were estimated by fitting the observed rates as a function of dCTP concentration to a hyperbola (Equation 2, see “Experimental Procedures”), k₃ = 18 ± 1 s^-1, k_-3 = 21 ± 1 s^-1, and K_d,dCTP = 210 ± 46 μm. C, the maximal amplitude reached at each concentration of dCTP in A was plotted and fit to a hyperbola to obtain an equilibrium dissociation constant, K_eq(G) = 41 ± 7 μm.

FIGURE 6.

Kinetic scheme of steps prior to phosphodiester bond formation. dNTP dissociation constants and conformational change rates were based on Dpo4 T239W fluorescence measurements. E, enzyme (Dpo4); E^*, enzyme conformation posed for catalysis. A,dCTP opposite a template G; B, opposite 8-oxoG (vide infra). See Figs. 5 and 11.

FIGURE 7.

Kinetics of fluorescence decay following phosphodiester bond formation. Initial time points (i.e. Fig. 3) were excluded to show the fit of the data to a single decay exponential (Equation 3, see “Experimental Procedures”). A, Dpo4 T239W·DNA_G (1 μm) was mixed with dCTP: 15 μm (red), 30 μm (orange), 50 μm (green), 120 μm (blue), 240 μm (purple), and 500 μm (brown), and the resulting fluorescence changes were recorded. Inset: decay rates plotted as a function of dCTP concentration.

FIGURE 8.

Comparison of rate of PP_i release with incorporation of C opposite G. Dpo4 T239W (1.2 μm) was preincubated with 200 μm DNA_G prior to rapid mixing in the stopped-flow apparatus with 500 μm dCTP and 1.1 μm PBP-MDCC with 0.005 unit of PPase ml^-1, 5 mm MgCl₂, and a phosphate mop (200 μm N⁷-MeGuo and 0.2 unit of PNPase ml^-1) present in both syringes. PP_i dissociation was monitored by stopped-flow fluorescence measurements upon binding of the released P_i to PBP-MDCC, k = 2.1 s^-1 (the residuals trace is shown in the inset). Superimposed are data from rapid quench experiments (○) monitoring product formation using a ³²P-labeled primer under identical conditions.

FIGURE 9.

Fitting of pre-steady-state and steady-state rates for dCTP polymerization opposite a template G by Dpo4 T239W to a minimal kinetic model. See supplemental Figs. S4-S6 for data, script, and result files. A, minimal model, with rate constants. E, Dpo4 T239W; D_n, substrate DNA; D_n + 1, product DNA; and E^*, Dpo4 T239W in active conformation. B, plot of the rate of phosphodiester bond formation by Dpo4 T239W k_obs values (•) versus dCTP concentration (Fig. S1B) by fitting to the minimal model (solid line). C, model fit to plots of product formation (•) versus time dependence on dCTP concentration (Fig. S1A). D, model fit to steady-state plot of v (k_obs) (•) versus dCTP concentration plot (Fig. S1C). The lines (C and D) were fit to the rate constants in the model (A) using the program DynaFit. See supplemental Figs. S4-S6 for DynaFit scripts and results.

FIGURE 10.

Kinetic model with an additional step representing a dissociated active complex. A, adjusted minimal model (Fig. 9A) with the added step E^*·DNA_G·dCTP Δ E^*·DNA_G + dCTP. B, model fit (solid line) to plots of product formation (•) versus time at various dCTP concentrations (Fig. 9C), with the added step. The model was also fit to a steady-state plot of v versus dCTP concentration with a result virtually identical to that in Fig. 9D (results not shown).

FIGURE 11.

Fluorescence changes observed upon DNA release from Dpo4 T239W and nucleotide binding to a Dpo4 T239W·DNA^dd complex with an 8-oxoG template base. A, the experiment of Fig. 4A was repeated, and fluorescence changes were recorded upon release of from Dpo4 T239W, k_off = 17 ± 3 s^-1. B, fluorescence changes induced by addition of dCTP opposite the template containing 8-oxoG: 5 μm (black), 15 μm (red), 30 μm (orange), 50 μm (green), 120 μm (blue), 240 μm (violet), and 500 μm (brown). C, rates of dCTP-induced fluorescence changes and ground state K_d,dCTP opposite template 8-oxoG were estimated, k₃ = 34 ± 2.5 s^-1, k_-3 = 4.5 ± 0.9 s^-1, and K_d,dCTP = 170 ± 42 μm. D, the maximal amplitudes measured in C were plotted and fit to a hyperbola to obtain an estimate of the equilibrium dissociation constant of dCTP bound opposite 8-oxoG, K_eq(G^*) = 11 ± 1 μm.