Independent and Stochastic Action of DNA Polymerases in the Replisome

Abstract

It has been assumed that DNA synthesis by the leading- and lagging-strand polymerases in the replisome must be coordinated to avoid the formation of significant gaps in the nascent strands. Using real-time single-molecule analysis, we establish that leading- and lagging-strand DNA polymerases function independently within a single replisome. Although average rates of DNA synthesis on leading and lagging strands are similar, individual trajectories of both DNA polymerases display stochastically switchable rates of synthesis interspersed with distinct pauses. DNA unwinding by the replicative helicase may continue during such pauses, but a self-governing mechanism, where helicase speed is reduced by ∼80%, permits recoupling of polymerase to helicase. These features imply a more dynamic, kinetically discontinuous replication process, wherein contacts within the replisome are continually broken and reformed. We conclude that the stochastic behavior of replisome components ensures complete DNA duplication without requiring coordination of leading- and lagging-strand synthesis. PAPERCLIP.

Keywords: DNA polymerase; DNA replication; replication fork coordination; replication fork progression; single-molecule analysis.

Figure 1. Visualizing leading- and lagging-strand synthesis using a rolling-circle single-molecule assay

A. Schematic of TIRF microscope and flow-channel. B. Side-on view, showing surface-attached DNA replication products. C. Cartoon showing assembly and live visualization of replication. dsDNA is visualized with SYTOX Orange fluorescent stain. D. Micrograph showing products live, ~180 s from start; three extending molecules identified. Scale bar: 10 μm, equal to 37.0 kb dsDNA at 2,500 μl/h (Figure S1). E. Time-lapse of three replicating molecules from D, showing synthesis with time. F. Kymographs of molecules from D, showing linear fits to trajectories yielding average rates of replication fork progression. Arrowheads: non-replicating substrates. G. Histogram of replication rates; mean rate, 470 ± 180 bp•s⁻¹ (molecules, n=84), from Gaussian fit.

Figure 2. Individual replication fork progression is independent of primase

A Micrographs showing replication products at 10 min where: i, all components present; or a component omitted: ii, DnaB and DnaC810; iii, Pol III*; iv, β; v, SSB; vi, primase. Composite, false-colored fields show anchor points for molecules that contain ssDNA, except i or v, where only long products were seen. In vi, surfaces were sparsely populated with DNA to avoid any ambiguity in molecule identification. Cyan, fields with flow off; magenta, same field with flow on showing fully-extended molecules. Molecules are bracketed for clarity. Scale bar: 10 μm, equal to 33.9 kb dsDNA or 80.3 knt SSB-bound ssDNA at 4,000 μl/h, without Mg²⁺ under end-point conditions (Figure S1). B. Cartoon showing leading strand only product in a reaction lacking primase. C. Composite, false-colored image showing leading strand only replication without primase. Three replicating molecules (1, 2, 3) are identified with brackets. Image shows motion of the SYTOX Orange-stained circular template across the field. The field is composed of seven snapshots at 50-sec intervals, colored red through violet (see legend). Scale bar: 10 μm, equal to 105 knt ssDNA•SSB at 2,500 μl/h under live conditions (Figure S1). *, spurious priming event (see also Movie 2). Molecules a, b and c are referred to later. D. Time-lapse, at 50-second intervals, of Molecules 1, 2 and 3 identified in C, colored by time-point as per C. E. Kymographs of molecules, numbered per C and D, showing fork progression without primase. Dashed grey line: position of anchor. Linear fits are from initiation to termination, yielding average fork rates. Pauses are included in the average here. F. Histograms of fork progression rates in the presence (grey) and absence of primase (light blue). Histograms fit to single Gaussians (R²: with primase, 0.80; without primase, 0.94); no outliers were rejected. n, molecules. G. Processivities of single replisomes from live imaging experiments. Whisker plots of molecule lengths, with (320 nM) or without primase and/or β in flow. Data from 2 (primase, no β) or 3 (others) experiments. Horizontal bars, median; vertical bars, interquartile range. ***, significantly different pairs of populations (Kruskal-Wallis; P < 0.05); other pairs not significantly different.

Figure 3. Leading-strand polymerization is kinetically discontinuous

A. Correlation of leading strand only synthesis pauses with duplex unwinding. (i) Plots of template displacement against time for molecules a, b and c (Figure 2C) replicating without primase. Data were fit to multi-segment lines (blue lines), yielding rates of synthesis (nt•s⁻¹), pause times and positions, and the lengths of synthesis bursts between pauses. (ii) Kymographs of the molecules in (i). (iii) Determination of DNA unwinding rates during pauses in synthesis. Sections of monotonic unwinding fit with straight lines (orange; rates in bp•s⁻¹), using pauses (i) as points of inflection. Magenta dotted lines: fully base-paired template (8,644 bp). B–E. Histograms of (B) burst rates of leading-strand synthesis, (C) run lengths of bursts between pauses, (D) pause times between bursts, and (E) DNA unwinding rates, determined without primase, and in the presence (yellow) or absence (grey) of β. Data from 5 (+β) or 3 (−β) experiments and n observations. Means from single or double Gaussian fits ± S.D. (R²: B, +β, 0.97; −β, 0.97; E, +β, 0.97, excluding outliers > 130 bp•s⁻¹; −β, 0.87). Data in D fit to single exponential (+β, τ ~ 12 s, R²=0.99; −β, τ ~ 15 s; R²=0.96), ignoring the under-sampled first bin. n, trajectories = 100.

Figure 4. Leading- and lagging-strand polymerases function autonomously

A. Reaction schematic, with experiments performed under low flow and products examined under high flow at a defined end-point. B. Micrographs of flow-extended products: dsDNA stained by SYTOX Orange; black gaps are ssDNA•SSB. Micrographs are false-colored magenta or cyan per whether flow was pulsed on or off (no primase and 2 nM primase), respectively; molecules are bracketed for clarity. Cartoons (white) show interpretations of dsDNA and ssDNA•SSB tracts for one molecule per panel. C, D. Plots of median (horizontal bars) and interquartile range (vertical bars) of (C) total dsDNA and (D) total gaps, per molecule, for range of primase concentrations (≥3 replicates). n, total number of molecules. E. Plot of fraction of total lagging-strand synthesis per total leading-strand synthesis versus primase concentration; insert shows zoom (replicates, N ≥ 3). Data fit to a rectangular hyperbola: K_½ = 9.3 ± 0.9 nM (S.E.). F, G. Plots of median (horizontal bars) and interquartile range (vertical bars) of (F) individual dsDNA tract lengths and (G) individual ssDNA•SSB lengths from N ≥ 3 replicates, for range of primase concentrations. n, number of molecules observed per condition. *, rare spurious priming events observed at 0 nM primase (n=8). H. Plot of dsDNA and ssDNA•SSB tract lengths versus primase concentration, expressed as the mean of population means (replicates, N≥3, ± S.E.M.).

Figure 5. Visualization of Okazaki fragment termini shows a direct relationship between primase concentration and priming frequency

A. Cartoon (top) showing method used to label 3′-ends of OFs ends (red wavy lines; middle). Cartoon (bottom) shows expected product when stained with SYTOX Orange and labeled with anti-digoxigenin (Figure S5): magenta, dsDNA tracts; green, OF 3′ termini; blue, anchor points; OF, PD: Okazaki fragment length, priming distance; defined in text. B. Representative false-colored micrographs with 4 to 320 nM primase in flow. dsDNA (magenta), OF ends (green) and merged images are shown for each field. Molecules are bracketed for clarity. One molecule in each field is highlighted and expanded in (C). C. Five magnified molecules from (B; a thru e). D. Semi-log plot of Okazaki fragment length against primase concentration. Population means ± 95% confidence intervals (bars); replicates, N=1 for ≥2 nM primase); N=2 for 1 nM primase. E. Primer utilization (reciprocal of priming distance) plotted against primase concentration. Data fit to rectangular hyperbola: K_M,app = 17 ± 3 nM (S.E.). Error bars: reciprocal of interquartile range of priming distance.

Figure 6. Lagging-strand synthesis occurs at the same burst rate and processivity as leading-strand synthesis

A. Plot of mean number (± 95% C.I.) of digoxigenin-labeled 3′ foci detected per dsDNA tract (Figure 5), versus primase concentration. Inset: expanded view, showing limit at unity (dashed red line). B. Histograms of the number of digoxigenin-labeled 3′ foci detected, as a function of primase concentration. n, number of molecules. C. Histogram showing distribution of Okazaki fragment lengths from end-product data at 1 nM and 2 nM (replicates, N=3 per condition; n=422), assuming one OF per tract. OFs synthesized up to the anchor point were rejected (see text). Only molecules with one OF were considered. D. Live replication experiment with 1 nM primase in flow. Image is a false-colored composite of the same field under flow at 0 s (red) and 400 s (grayscale). Three molecules undergoing OF synthesis identified with solid brackets. A molecule that undergoes only leading-strand synthesis is identified with a dotted bracket. E. Waterfall plot showing change in fluorescence along a 1D line profile drawn across Molecule 1 in (D) over 400 s. Cartoons show interpretation. Dotted ellipse shows a second molecule ignored for the analysis of Molecule 1. F. Time-lapse of Molecule 1 from (D), showing Okazaki fragment synthesis (x) and leading-strand-only synthesis (y). Graphs (right) show the growth of the OF (top, red, x) and leading strand synthesis (bottom, blue, y). Data fit to segmented lines to yield rates (± S.E. of fit). G. Graph showing synthesis of Okazaki fragments for the three molecules in D. Data fit to segmented lines, with both pauses and end-points constrained to zero rate, yielding indicated rates (± S.E. of fit). H. Histogram of lagging-strand burst rates (between pauses) from n=39 (five experiments; 24 molecules), fit to a single Gaussian to determine population mean ± SD.