Mechanochemical regulations of RPA's binding to ssDNA

Abstract

Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA.

Figure 1. AFM images of RPA-bound ssDNA.

(A). Randomly coiled naked M13 ssDNA. (B–D). RPA-bound ssDNA formed in different RPA to ssDNA stoichiometric ratios: 1:50 (B), 1:10 (C) and 1:1 (D). DNA concentrations in (A–D) were kept constant.

Figure 2. Concentration dependent dynamics of RPA's ssDNA binding.

(A). A single 576 bp dsDNA is tethered between coverglass and paramagnetic bead surfaces through the two ends on the same DNA strand (I). By stretching the tethered DNA strand at ~ 65 pN, the untethered strand is peeled off (II), leaving an ssDNA strand under force (III). (B). Binding dynamics of RPA to ssDNA upon various protein concentrations from 1 nM–1 μM are introduced at a constant force of 7.4 pN smoothed with 30-points (0.3-second data) FFT filtering. (C). RPA concentration dependent DNA extension elongation Δz_max (tri-angles), which is the difference between the steady state extension (average extension in the last 100 seconds in Fig. 2B) and the naked ssDNA extension before introduction of RPA. Error bar for each symbol is the standard error (s.e.) from multiple (>3) independent measurements.

Figure 3

(A). Force-extension curves of RPA-bound ssDNA formed with varying RPA concentrations. Inset shows the fitting of experimental data in the force range of 7–45 pN by the Marko-Siggia formula (R²>96%), which gives the effective bending persistence length (A_eff) and effective contour length (L_eff) at each protein concentration (B). From the RPA dependent A_eff, the RPA occupation fraction on ssDNA at different proteins concentrations are calculated and shown in (C). The error bars in (A) are standard deviation (s.d.) from multiple (>3) force-scans at each condition. The error bars in (B–C) are standard errors (s.e.) from multiple ssDNA tethers. The black dash line in (C) is average of Hill equation fitting of data obtained from multiple independent tethers (R² > 91%). (D). Monovalent salt dependence of dissociation constant (solid circles) and Hill coefficient (Hollow circles, inset). The gray error bars indicate standard deviation (s.d.) and black error bars indicate standard errors (s.e.) from multiple (>7) ssDNA tethers.

Figure 4

(A). Comparison of the binding dynamics of 1 μM RPA to ssDNA in the absence (black) and presence (red) of zinc under constant force of 7.4 pN, showing a different steady state extension. (B). Comparison of the steady state force-extension curves between zinc-free (wine) and zinc-induced (olive) RPA-bound ssDNA formed after saturation binding at 7.4 pN. Data indicated by solid and hollow symbols are obtained during force-decrease and force-increase scans, respectively. Force-extension curve of bare ssDNA (black) is plotted for comparison. Inset show the quantification of the stiffness of ssDNA and RPA-bound ssDNA. (C). Extension time trace of RPA-bound ssDNA at constant forces in the presence of zinc. Inset shows the zoom in of extension increase of the initial 70 s after force jumped from 4 pN to 43 pN.

Figure 5. Schematics of a model that describes the effect of force on the conformation of RPA-coated ssDNA.

In the absence of force, RPA ssDNA binding domains bind distant ssDNA sites, stabilizing condensed ssDNA. In contrast, in the presence of force, RPA binds to the force extended ssDNA conformation, resulting in an ordered extended conformation with increased bending rigidity.