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. 2007 Jul 31;104(31):12610-5.
doi: 10.1073/pnas.0700920104. Epub 2007 Jul 18.

Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase

Guobin Luo  1 , Mina Wang, William H Konigsberg, X Sunney Xie
Affiliations

Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase

Guobin Luo et al. Proc Natl Acad Sci U S A. .

Abstract

We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Kinetic scheme and conformational change of DNA pols. (A) Minimum kinetic scheme shared by different DNA pols. To extend a primer, the following steps are required: Step 1, binding of DNA to form a binary complex; Step 2, binding of a dNTP substrate to form a ternary complex; Step 3, a series of conformational changes in the ternary complex that ensure proper alignment of the reactants; Step 4, phosphoryl transfer; Step 5, another conformational change that relaxes the complex; Step 6, pyrophosphate release and translocation of the DNA, bringing the P/T into a position poised for the next synthetic step. This scheme was adapted from ref. . The states accessible in our fluorescence assay are shown within the dashed rectangle. (B) Conformational change of T7 pol from the open form (gray; PDB entry 1SL0; ref. 7) to the closed form (in color; PDB entry 1T7P; ref. 7). Two structures are superimposed by aligning the polymerase palm domains. The conformational change occurs through coordination of Mg2+ (pink) by both the triphosphate moiety of the dNTP (blue) and two conserved residues with carboxylic side chains (red).
Fig. 2.
Fig. 2.
Experimental setup for our single-molecule fluorescence observations. Cy3-P/T was immobilized on the surface of a glass coverslip through a biotin–streptavidin linkage. DNA pol and dNTP were supplied in the solution above the coverslip. TIR, total internal reflection.
Fig. 3.
Fig. 3.
Detection of T7 pol binding by Cy3 fluorescence. (A) Fluorescence intensity trajectory of a surface-immobilized Cy3-P/T duplex. The arrow marks the time when the polymerase solution was flowed in. Fluorescence intensity trajectories shown here were all filtered with a nonlinear filter (32). (B) Histograms of the durations of the polymerase on and off states, with exponential fits (blue curves). (C) Estimation of the Cy3 position in the T7 pol binary complex. The base (red) corresponds to the second to last base from the 5′ end of the template (yellow) where Cy3 is attached. The residues deleted from the wt T7 pol in the Sequenase are shown in blue. In the binary complex, Cy3 should be located between the base (red) and the residues (blue). The circle shows the approximate position of Cy3. (D) Cy3 structure (Inset) and the potential energy surface of Cy3, adapted from ref. . The excited-state process following the dashed arrows is a nonradiative relaxation pathway sensitive to local environment.
Fig. 4.
Fig. 4.
Conformational change of T7 pol upon dCTP binding detected by Cy3 fluorescence change. (A) Fluorescence intensity trajectories of the Cy3-ddP/T duplex in a mixture containing 10 nM T7 pol, 20 μM ddATP, and 0–5 μM dCTP in 50 mM Tris buffer, pH 7.5, with 10 mM MgCl2. (B) Part of the fluorescence intensity trajectory of the duplex Cy3-ddP/T with an expanded y axis, obtained with 10 nM T7 pol and 50 nM dCTP. The fluorescence intensity histogram, along with a fit of Gaussian peaks on the right, clearly shows three intensity levels. (C) Statistics of the lifetimes of the T7 pol binary and ternary states with 50 nM of dCTP, with exponential fits (blue curves).
Fig. 5.
Fig. 5.
Ensemble-averaged measurements of the conformational change of T7 pol complex upon dCTP binding. (A) Stopped-flow fluorescence intensity traces of the Cy3 probe after mixing a solution of Cy3-ddP/T·T7 pol complex with a solution of different concentrations of dCTP. The black dashed lines represent double exponential fits of the data. (B) Experimental setup for continuous-flow measurement based on hydrodynamic focusing. (C) Fluorescence intensity traces of the Cy3-ddP/T·T7 pol after mixing with dCTP, obtained with a continuous-flow device. The black dashed lines are single exponential fits of the data. (D) dCTP concentration dependence of the observed rates of conformational change. A Michaelis–Menten fit was used to extract the rate of conformational change and Kd value for this process.
Fig. 6.
Fig. 6.
Fluorescence intensity trajectories of the Cy3-ddP/T duplex showing a slow T7 pol conformational change induced in the presence of 0.25 mM noncomplementary substrates (dTTP or dGTP).
Fig. 7.
Fig. 7.
Free-energy diagram for complementary and noncomplementary dNTP substrate binding (calculated from previous reports) and for the open-to-closed conformational change of the T7 pol ternary complex (calculated from our experimental data).
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