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. 2012 Jul;40(13):6174-86.
doi: 10.1093/nar/gks253. Epub 2012 Mar 20.

Mechanism of strand displacement synthesis by DNA replicative polymerases

Maria Manosas  1 , Michelle M Spiering, Fangyuan Ding, David Bensimon, Jean-François Allemand, Stephen J Benkovic, Vincent Croquette
Affiliations

Mechanism of strand displacement synthesis by DNA replicative polymerases

Maria Manosas et al. Nucleic Acids Res. 2012 Jul.

Abstract

Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.

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Figures

Figure 1.
Figure 1.
Observation of strand displacement synthesis and primer extension by single holoenzymes. (A) Schematic representation of the experimental set-up. A DNA hairpin substrate was tethered between the glass surface and a magnetic bead held in a magnetic tweezers. A holoenzyme loaded at the 3′ end of the primer may display strand displacement synthesis activity (light green), exonuclease activity (yellow) or primer extension activity (dark green). Experimental traces corresponding to wt T4 holoenzyme activity (B), exo-T4 holoenzyme activity (C) and wt T7 holoenzyme activity (D) are shown. The total change in extension observed in the two phases correspond to the polymerization of 1200 nt. The right axis shows the extension of the replicated substrate as computed in Supplementary Figure S2.
Figure 2.
Figure 2.
Strand displacement activity of the wt T4 holoenzyme. (A) Schematic representation of the experimental protocol. Primer synthesis was initiated with the hairpin open at Fopen = 16 pN; the force was then lowered to various Ftest values and strand displacement activity was observed. (B) Traces of strand displacement activity recorded at Ftest values of 13 pN (blue), 11 pN (red), 8.5 pN (green) and 5 pN (magenta). Bars show the extension change corresponding to the synthesis or degradation of 100 nt. (C) Velocity distributions of instantaneous enzyme rates measured during strand displacement activity at different forces (colours as in panel B). The number of molecules (Nmol) analysed was 9, 14, 21 and 15 for 13, 11, 8.5 and 5 pN cases resulting in 82, 131, 115 and 224 number of enzymatic traces (N), respectively. (D) The mean synthesis rate formula image (light green), mean degradation rate formula image (orange) and mean strand displacement rate formula image (purple) measured for the wt T4 holoenzyme shown as a function of the applied force. Error bars are the s.e.m. The number of molecules and enzymatic traces analysed varies between Nmol = 9–27 and N = 75–253, respectively, depending on the condition.
Figure 3.
Figure 3.
Analysis of the pauses observed during wt T4 holoenzyme strand displacement activity. (A) An experimental trace indicating the assignment of the elongation branch (regions in green) and the degradation branch (regions in orange). Pauses during elongation and degradation are shown in dark green and brown, respectively, while the transition time from pol to exo and from exo to pol are shown in red and magenta, respectively. The bar shows the extension change corresponding to the synthesis of 100 nt. (B) Distributions of the residence pause times during the elongation (Number of events Ne = 221), and degradation (Ne = 55) branches, and the transition times for switching between pol and exo (Ne = 173 and Ne = 153) fit to either a single- or double-exponential equation. The error bars are proportional to the inverse of the square root of the number of points for each individual bin. (C) Mean residence times, formula image, formula image, formula image and formula image shown as a function of the applied force. Errors are estimated from the single- or double-exponential fits.
Figure 4.
Figure 4.
Kinetic scheme for the wt T4 holoenzyme primer transfer reaction in strand displacement. (A) Diagram showing the effect of applied force to shift the equilibrium from the polymerization conformation (pol) to the exonuclease conformation (exo) through an inactive intermediate (I) during strand displacement synthesis. An off-pathway intermediate (ip) and unbound polymerase state (D) are also present in the scheme. For each transition d and n correspond to the distance change in the molecular extension of the DNA in nm and the number of unwound or annealed bases, respectively, associated with the conformational transition. (B) State frequencies as defined in Equation (1) shown as a function of the applied force and fit to a single-exponential equation. The distance d and the corresponding estimation of bases unwound or annealed, n, are obtained from the fits to Equation (2).
Figure 5.
Figure 5.
Predictions from the kinetic model. (A) Kinetic rates (as shown in Figure 4A) estimated from the measurements of the state frequencies and mean lifetime of each state using Equation (S11). Error bars are obtained by propagating the errors associated with the mean lifetime and state frequencies. (B) A comparison of the measured mean strand displacement rate for the wt T4 holoenzyme (purple squares) to the predicted rates for the elongation (green line) and degradation (orange line) branches as estimated from Equations (S7) and (S9) and for the whole kinetic scheme including the two branches (magenta line) as estimated from Equation (S4) shown as a function of the applied force. Error bars are the s.e.m.
Figure 6.
Figure 6.
Cyclic polymerase assay and application for single-molecule Sanger sequencing. Schematics (left panels) and experimental traces (right panels) of the CPA on the 1.2 Kbp long DNA hairpin (LH) substrate (A) and 100 bp short DNA hairpin (SH) substrate with LNA block (B). Synthesis (green) took place at higher applied forces (Ftest) until the LNA block (purple) was reached or the force was decreased to Fexo inducing the exonuclease activity (orange) and recovering the initial hairpin. (C) Utilizing the CPA for single-molecule Sanger sequencing, a trace recorded during a single cycle displayed transient pauses due to the low dATP concentration, as well as a permanent arrest due to the incorporation of ddATP (left panel). Distribution of the frequency of enzyme arrests corresponded with the expected position of adenosine nucleotides in the known DNA test sequence (right panel, N = 53). Bars show the extension change corresponding to the synthesis or degradation of 100 nt and 10 nt for the LH and SH results, respectively.
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