Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

Abstract

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 11 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.

Fig. 1.

Labeled T7 DNA polymerase retains primer extension activity and diffuses along the length of λ-phage DNA. (A) Amount of dTMP incorporated as T7 DNA polymerase extends a primer annealed to circular, single-stranded M13 DNA is dependent on protein concentration. Labeled and unlabeled gp5/trx show nearly identical activity. Labeled and unlabeled gp5 also have similar activity, but are substantially less active than gp5/trx due to the absence of thioredoxin. (B) Kymographs of gp5/trx complexes diffusing along DNA. Each kymograph represents 222 exposures of 30 msec each. The direction of flow is down. (C) Trajectories of the gp5/trx complexes obtained from the kymographs shown in (B). (D) MSD vs. time calculated from the trajectories in (B) and (C). Solid circles correspond to the solid line trajectory and open circles correspond to the dotted trajectory. The first ten data points are fit with a linear dependence and the slope was used to determine the linear diffusion coefficient.

Fig. 2.

Observed diffusion coefficients and net displacements. Histograms of observed diffusion coefficients, plotted on a log scale (Left), and net displacements, plotted on a linear scale (Right). The total displacement from the start of each trajectory to its end is normalized for the length of the trajectory. Positive displacement is in the direction of the buffer flow. Each histogram consists of data collected under identical conditions. (A) For gp5/trx, the diffusion coefficient distribution remains essentially unchanged over all experimental conditions. Net displacement distributions are symmetric, centered near zero, and unaffected by the ionic strength of the experimental buffer. (B) For gp5, in the absence of thioredoxin the diffusion coefficient distribution shifts toward higher values as the ionic strength of the experimental buffer increases. The normalized net displacement distributions are skewed in the direction of buffer flow, with the mean value increasing with the ionic strength of the experimental buffer. (C) The distributions of the observed diffusion coefficients and net displacements of gp5 + thioredoxin at a buffer ionic strength of 64 mM are most similar to those of gp5/trx.

Fig. 3.

Salt dependence of diffusion coefficient and flow-induced drift. (A) In the absence of thioredoxin, the diffusion coefficient of gp5 increases with the ionic strength of the experimental buffer; gp5/trx diffusion is insensitive to changes in experimental buffer composition. Data points and error bars represent the geometric mean and standard error of the geometric mean, respectively, and are calculated as described in SI Text. (B) The mean normalized net displacement indicates the amount of flow-induced drift in the diffusion of molecules along the DNA. As the ionic strength of the buffer increases, gp5 trajectories increasingly drift in the direction of the buffer flow. Trajectories of gp5/trx show minimal drift that remains constant with increasing buffer ionic strength. Data points and error bars represent the mean and the SEM, respectively. (C) Summary of data at a buffer ionic strength of 64 mM for gp5/trx, gp5 alone, and gp5 + thioredoxin. Light colored bars represent the mean diffusion coefficient observed, and darker colored bars represent the flow-induced drift for each protein. Error bars correspond to the SEM. In the absence of thioredoxin, gp5 has a significantly higher mean diffusion coefficient, and greater degree of flow-induced drift. When an excess of unlabeled thioredoxin is added to the experiment, the diffusion coefficient and flow-induced drift of gp5 both revert to values indistinguishable from those observed for labeled gp5/trx.

Fig. 4.

T7 DNA polymerase binds along the length of λ-phage DNA. (A) At a constant protein concentration the amount of fluorescently labeled protein bound to a single, tethered DNA molecule decreases as the concentration of monovalent cations in the experimental buffer increases. Each panel contains a view of gp5/trx binding to the same DNA molecule at a different monovalent cation concentration (Left). Experiments performed with gp5 in the absence of thioredoxin yielded similar results. (B) Log–log scaled plots of the observed intensity per unit length of DNA vs. the total monovalent cation concentration for gp5/trx. Each data point (open circles) represents the average intensity per unit length for one DNA molecule in one experimental condition; linear fits through all the data obtained for a single DNA molecule are shown in gray. The thick black line is defined by the means of the fit parameters for all the DNA molecules, and has a slope of -4.34 (± 0.26 SEM), indicating five charge–charge interactions between the protein and the DNA backbone. (C) Log–log scaled plots of the observed intensity per unit length of DNA vs. the total monovalent cation concentration for the gp5 free of thioredoxin, colored as in (B). In this case, the mean fit has a slope of -3.29 (± 0.26 SEM), indicating four charge–charge interactions between the protein and the DNA backbone.