Conformational dynamics of bacteriophage T7 DNA polymerase and its processivity factor, Escherichia coli thioredoxin

Abstract

Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.

Fig. 1.

Crystal structure of T7 gp5/trx bound to primer template and an incoming nucleotide. Gp5 is shown in blue and trx in brown. The DNA is shown in orange. Trx binds to the unique 76-residue segment at the tip of the thumb, trx-binding domain (TBD). [PDB ID code 1T8E (23)].

Fig. 2.

Effect of ionic strength on polymerase activity and on the affinity of the polymerase for a primer template. Polymerase activity (A) was measured in the DNA polymerase assay containing 40 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 50 mM potassium glutamate, 0.25 mM dTTP, dCTP, dGTP, and [α-³²P] dATP, 20 nM primed M13 DNA, and 5 nM gp5 in the absence of trx (blue) or in the presence of 25 nM trx (red), at the indicated concentrations of NaCl. After incubation at 37 °C for 10 min, the amount of [α-³²P] dAMP incorporated into DNA was measured. (B) The binding of gp5 and gp5/trx to a primer template in the presence of the incoming dNTP (see Inset) was measured using a nitrocellulose filter binding assay. The reaction contained 40 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 50 mM potassium glutamate, 250 μM ddATP and ddGTP, and 2.5 μM gp5 in the presence (red) or absence (blue) of 3 μM trx and 1 nM primer template. The primer template consisted of the primer (5′-CGAAAACGACGGCCAGTGCCA-3′ annealed to the radiolabeled template strand 5′-³²P (5′-CCCCTTGGCACTGGCCGTCGTTTTCG-3′). The 3′-terminal nucleotide of the primer is ddCMP. The reaction mixture was loaded onto a nitrocellulose membrane laid atop a Zeta-probe membrane (see Materials and Methods) fixed on a dot microfiltration apparatus. The protein-DNA complex bound to the nitrocellulose membrane, and the free DNA on the Zeta-probe membrane were measured. (C) The distribution of electrostatic surface potentials for gp5/trx (7) calculated using the Adaptive Poisson-Boltzmann Solver plug-in (27) embedded in PyMol (www.pymol.org). The values of surface potentials are shown as a spectrum ranging from the negative value -4kT (red) to the positive value +4kT (blue). The residues in the DNA binding cleft that contact DNA are represented by yellow dots. Two different views of gp5/trx are presented.

Fig. 3.

Single-molecule analysis of leading-strand synthesis mediated by gp5/trx or gp5 in the presence of gp4 helicase. (A) Experimental design. Duplex λ DNA (48.5 kb) is attached to the surface of the flow cell via the 5′ end of the fork using biotin-streptavidin interaction, and the 3′ end is attached to a paramagnetic bead using digoxigenin-anti-digoxigenin interaction. The replication reaction in the flow cell contains 20 nM gp4 (hexameric concentration), 20 nM gp5/trx, or 100 nM gp5 in a buffer consisting of 600 μM each of dATP, dTTP, dCTP, and dGTP, 10 mM DTT, and 10 mM MgCl₂. DNA synthesis by proteins leads to conversion of the dsDNA to ssDNA, resulting in shortening of the DNA ligand that was accompanied by the movement of the bead against the direction of the flow. (B) Rate and processivity of leading-strand synthesis by gp5/trx or gp5 in the presence of gp4 helicase. Examples of single-molecule trajectories for leading-strand synthesis are shown. Rate and processivity were calculated by fitting the distributions of individual single-molecule trajectories using Gaussian and exponential decay distributions, respectively. Twenty-two single events were used to calculate rate and processivity for gp5/trx, and 11 events for gp5. Standard errors represent the accuracy in fitting of these distributions.

Fig. 4.

SAXS data and low-resolution solution structure of gp5 and gp5/trx. (A) Low-angle data (0.0088–0.38 Å^-1) were obtained at a detector length of 2 m. (B) Distance distribution function p(r) of free gp5 (blue) and gp5 upon binding to trx (red) with D_max of 80 Å and 105 Å, respectively. p(r) was obtained using the computer program GNOM (18). The contribution of gp5 to the structure of gp5/trx was obtained by subtraction of the trx theoretical spectrum (PDB ID code 2trx, depicted in gray) from the gp5/trx theoretical spectrum (depicted in light dashed gray). (C) The corresponding ab initio model of gp5, obtained using the computer program GASBOR (20), is presented at Left. The gp5 contribution to the crystal structure of gp5/trx (PDB ID code 1T8E) is presented at Right.

Fig. 5.

Footprint of gp5 and gp5/trx on a primer template. (A) Schematic representation of gp5/trx bound to a primer template. The primer template used in this analysis consisted of a 5′-³²P radiolabeled 26-nt template annealed to a 21-nt primer strand such that there are five 5′-nucleotides of ssDNA on the template. ddATP and ddGTP are present in the reaction. Upon addition of DNA polymerase ddAMP is incorporated resulting in chain termination, and the next incoming nucleotide ddGTP cannot be incorporated but locks the polymerase onto the primer template. Gp5/trx has been superimposed on the DNA to create a model for the interpretation of the results. (B) Protection of the template strand from digestion by exonuclease III. The DNA primer template (500 pM) was incubated with 1 μM gp5 or gp5/trx. Reaction mixtures contained ddATP and ddGTP (250 μM). Exonuclease III (10 units) was added and the reaction was incubated at 37 °C for 10 min. The radiolabeled products were separated on 25% polyacrylamide gel containing 3 M urea and visualized by autoradiography.

Fig. 6.

Normal mode analysis (NMA) of gp5. NMA analysis (Web site: http://lorentz.immstr.pasteur.fr/gromacs/nma_submission.php) was carried out with the crystal structure of T7 DNA polymerase (PDB ID code 1T8E). The conformational changes of gp5 are depicted as obtained from NMA using 16 individual large domain-scale motions. The change in Cα conformation is presented.

Fig. 7.

Amino acids distribution in the DNA binding surface of T7 DNA polymerase and the large fragment of E. coli Pol I (Klenow fragment). The ratio between the type of amino acid (i.e., hydrophobic, negative, positive, or polar) and total number of amino acid residues constructing the DNA binding cleft of the enzymes is shown.