Figure 5. A3G binding to ssDNA/dsDNA hybrid construct.

(A) FECs of hybrid construct. A 6.5 kbp dsDNA construct is stretched using optical tweezers (blue diamonds). The first stretch follows the WLC model (yellow line), but a one knt section of ssDNA between two nicking sites dissociates during overstretching, altering the FEC of subsequent stretches (red triangles). At low forces, secondary structure compacts the ssDNA and the constructs total extension is consistent with the ssDNA section having zero net extension (magenta line). At higher forces, secondary structure is disrupted, and the ssDNA’s contribution to the construct’s total extension follows the FJC model (cyan line). (B) ssDNA extension change due to A3G binding at low force. The hybrid construct is held at a constant force and incubated with 50 nM A3G. At the lowest forces (4 pN in yellow and 8 pN in green), ssDNA is initially compacted due to secondary structure but increases it extension as A3G binds. (C) Average ssDNA extension changes. The average total extension changes (compared to initial extension, blue circles) and average relative extension changes (compared to FJC model, red triangles) are plotted as a function of force. Above ~10 pN, ssDNA extension is decreased similar to experiments using the fully ssDNA construct at 20 pN (green symbols). At lower forces ssDNA extension appears to increase, but when the initial decrease in extension due to ssDNA secondary structure prior to A3G incubation is taken into account, the final ssDNA-complex is still shorter than bare, linear ssDNA. (D) Average rate of extension change. While the timescale required for the ssDNA to reach an equilibrium length at forces above ~10 pN agrees with 20 pN data obtained using a fully ssDNA construct, this process is greatly slowed at low forces where significant ssDNA secondary structure is present. (E) Stabilization of ssDNA loops by A3G. After incubating the hybrid construct with A3G at near zero force so that the ssDNA region is completely unextended, the DNA is stretched at a rate of 100 nm/s. As compared to the compaction of ssDNA due to intrinsic secondary structure formation (panel B), much higher forces are required to extend the ssDNA once A3G is bound (blue line). This effect remains even after removing free A3G from solution (red line). Similar effects are seen when incubating with FW mutant A3G (green line), indicating this A3G looping does not require A3G oligomerization. Error bars are standard error based on multiple experimental replications (N ≥ 5) with different ssDNA molecules.

Figure 5—figure supplement 1. Kinetics and equilibrium of ssDNA secondary structure formation.

(A) When ssDNA extension is increased at a fast (red line) or slow (blue line) rate, the tension on the ssDNA increases and secondary structure is disrupted, resulting in a sudden increase in extension. Rapid increases in force result in the extension occurring at high forces. Conversely, on the return curves (dotted lines), secondary structure resulting in ssDNA compaction occurs in a rate-independent manner, and the ssDNA exhibits a consistent change in extension relative to its fully extended state. (B) Average ssDNA compaction as a function of force. Using return curves from different DNA constructs (N = 6), the average reduction in ssDNA extension due to secondary structure is calculated as a function of force (blue line). The fractional compaction (red line) is calculated by interpolating the ssDNA’s measured extension between the extension of the fully extended and compacted states.

Figure 5—figure supplement 2. A3G binding to dsDNA.

FECs of dsDNA in protein free buffer (black line) or in the presence of A3G (blue and red lines) overlap below the melting transition, indicating that A3G binding does not alter dsDNA polymer properties. The force required to overstretch dsDNA increases and binding to exposed ssDNA prevents reannealing of bases when the force is lowered (dashed lines).