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. 2012 May 22;109(21):8115-20.
doi: 10.1073/pnas.1204759109. Epub 2012 May 9.

Active DNA Unwinding Dynamics During Processive DNA Replication

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Free PMC article

Active DNA Unwinding Dynamics During Processive DNA Replication

José A Morin et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overview of experimental assay. (A) Schematic representation of the experimental design. A single DNA hairpin was tethered to functionalized beads inside a fluidics chamber. One strand of the hairpin (blue) is attached through a dsDNA handle to a bead held in the laser trap, while the complementary strand (red) is attached to a bead on top of a mobile micropipette (Methods). At a constant tension (f) the strand displacement and primer extension activities of the polymerase (red triangle) are detected as a change in distance between the beads, Δx1 and Δx2, respectively. (B) Representative replication traces of the wild-type (blue) and sdd mutant (green) polymerases showing in detail the distance changes during strand displacement (s.d., Δx1) and primer extension (p.e., Δx2) activities (f ∼ 11 pN). See Fig. S3 for additional experimental traces at different tensions.
Fig. 2.
Fig. 2.
Tension and sequence dependencies of the wild-type and sdd mutant rates. Only activities that replicate the full hairpin length were considered (Fig. S4A). (A) The tension dependency of the average strand displacement rate (Vsd) of the wild-type polymerase with and without pauses (full and empty blue circles, respectively) is well explained by our active model (solid blue line). (B) During strand displacement the wild-type polymerase spends longer times (blue, f ∼ 4 pN, N = 15) at the more stable hairpin positions (dotted lines). This behavior is well predicted by the proposed model (orange). For comparison, the experimental force-unzipping curve of the hairpin is shown in gray. (C) The different tension dependencies of the average strand displacement rate (Vsd) of the mutant with and without pauses (full and empty green circles, respectively) can be explained with the same active model when pause kinetics are included in (solid green line) or excluded from (dashed green line) the model. (D) Representative position versus time traces of the mutant polymerase during strand displacement. Identified pause events are shown in red, long pauses are located at the GC rich positions of the hairpin (gray lines). Insert shows the sdd mutant velocity distribution during strand displacement conditions (Fig. S5C). (A and C) For both polymerases, full and empty red circles represent the average primer extension rates (Vpe) with and without pauses. Solid red lines represent the sequence independent velocity value used in the model (128 nt/s). Error bars represent the standard error.
Fig. 3.
Fig. 3.
Pause kinetics of the wild-type polymerase. (A) Representative position versus time traces of the wild-type polymerase during strand displacement. Identified pause events are shown in red, gray lines show GC rich positions of the hairpin. Insert shows the wild-type velocity distribution during strand displacement conditions (Fig. S5A). (BD) Red dots represent primer extension and blue dots strand displacement conditions. (B) The pause length frequency distributions (s-2) can be fitted with a single exponential (f ∼ 5 pN). Primer extension, red line (R2 = 0.89, N = 22), strand displacement, blue line (R2 = 0.87, N = 38). (C) The pause frequency (s-1) decreases exponentially with tension (solid lines represent fit to data with equation 1) and (D) increases linearly with the GC content of the DNA (f ∼ 5 pN). Primer extension, red line (R2 = 0.81, N = 38), strand displacement, blue line (R2 = 0.78, N = 22). (E) Proposed kinetic model that explains the behavior of short-lived pauses (Short Pause 1). See Table 1 for rate values and the associated tension dependence, da1. For (B) and (D) error bars calculation is described in SI Text. For (C) error bars represent standard deviation.
Fig. 4.
Fig. 4.
Pause kinetics of the sdd mutant polymerase. (A) The pause length frequency distribution (s-2) during primer extension (f ∼ 6.5 pN, red dots) is compatible with a single exponential (solid red line R2 = 0.97, N = 25). For comparison the wild-type distribution from Fig. 3B is shown as a gray line. During strand displacement (f ∼ 6.5 pN, green dots) a double exponential is required to fit the data (green solid line, R2 = 0.93, N = 41). For (BD), green dots represent short pauses during strand displacement; red dots, short pauses primer extension; black dots, long pauses strand displacement. (B) The average length (seconds) of short pauses is tension independent while the average length of long pauses decreases exponentially with tension (black line fit to the data with Eq. 1). (C) For both pause types the pause frequency (s-1) decreases exponentially with tension (solid lines represent the fit to the data with Eq. 1) and (D) increases linearly with the GC content of the DNA (solid lines represent linear fits, R2 = 0.98, 0.99, 0.92). (E) Proposed kinetic model that explains the mutant pause behavior during strand displacement conditions. See Table 1 for rates and conformational changes values. For (A) and (D) error bars calculation is described in SI Text. For (B) and (C) error bars represent standard deviation.
Fig. 5.
Fig. 5.
DNA unwinding model. (A) Diagram showing notation used for modeling the polymerase movement during strand displacement conditions. M (red circle) defines the range of interaction between the polymerase and the junction; m is the number of ssDNA template nucleotides between the polymerase and the junction; l is the total number of replicated nucleotides and L is the full length of the hairpin. (B) Proposed DNA unwinding mechanism. Figures show the schematic representation of the Phi29 DNA polymerase with the DNA substrate during strand displacement (6). The protein is diagrammed in two levels. The upper level contains the exo (green), the TPR2 (blue) and thumb (orange) domains. The rest of the protein is shown as a gray circle. (1) and (2) Template bending at the TPR2-exo tunnel and steric exclusion of the complementary strand may generate mechanical stress at the fork junction promoting the unwinding of the first 2 bp (blue) of the fork. dNTP binding and hydrolysis fueled the polymerase forward movement. (3) For the sdd mutant, mechanical stress at the junction during unwinding of the more stable fork positions (where m = 0) could lead to destabilization of the template-tunnel interactions, favoring the entrance to the Long Pause 2 state. Fork destabilization by external tension (f) favors DNA unwinding, preventing the entrance to and promoting the exit from the inactive Long Pause 2 state. In (2) and (3) the initial position of the fork is shown in light gray.

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