Fig. 1: Single-molecule analysis of SSB’s effect on DNA replication.

A Experimental setup using dual-optical tweezers. DNA template (~8 kbp) with 25 nt overhang is tethered between optically trapped beads. High tension (45–50 pN) creates ssDNA template via exonuclease activity; reduced tension (10–20 pN) enables polymerization analysis. Inset shows T7 DNAp (PDB: 1T7P) and T7 gp2.5 (PDB: 1JE5) at the ssDNA/dsDNA junction. B Force-dependent changes in end-to-end distance. At 50 pN, exonuclease activity increases the distance. At 10–20 pN, SSB binding and polymerization decrease the distance. Transition from ssDNA (FJC model) to dsDNA (WLC model) enables activity quantification. Change-point detection algorithm determines polymerase/exonuclease activity shifts. C Representative replication trajectories with/without SSB at 10 and 20 pN. Dashed lines: reference rates without SSB. Black arrows: backtracking events. Trajectories include 3–5 s prior to tension transition, aligned to the transition time for comparison. D SSB effects on replication efficiency. At 10 pN: SSB significantly enhances efficiency from 180 ± 10 bp/s (N = 67) to 230 ± 20 bp/s (N = 72, p = 0.0467). At 20 pN: SSB reduces efficiency from 130 ± 10 bp/s (N = 63) to 80 ± 5 bp/s (N = 81, p < 0.0001). E Exonuclease binding probability analysis. Without SSB: higher probability at 20 pN (0.3 ± 0.05, N = 30) than 10 pN (0.2 ± 0.04, N = 35), indicating tension-dependent exo site binding. With SSB: probabilities decrease to 0.06 ± 0.02 (N = 47) at 10 pN and 0.3 ± 0.06 (N = 43) at 20 pN. Results show polymerase site preference. F SSB modulation of replication processivity under varying tensions. At 10 pN: SSB increases processivity from 720 ± 60 bp/event (N = 67) to 1400 ± 100 bp/event (N = 72, p < 0.0001). At 20 pN: decreases from 500 ± 40 bp/event (N = 63) to 380 ± 30 bp/event (N = 81, p = 0.0312). G Average pause duration analysis. Without SSB: decreases from 19 ± 2 s (10 pN, N = 61) to 13 ± 1 s (20 pN, N = 101), possibly due to hairpin impedance at lower force. With SSB: increases to 31 ± 2 s at 10 pN (N = 50, p < 0.0001), remains 15 ± 1 s at 20 pN (N = 99, p = 0.24). H Fluorescently labeled DNAp binding to ssDNA with/without SSB. Only the green channel was displayed. White arrows: DNAp trajectories. I DNAp coating density on ssDNA. Density calculated as the normalized difference between average photon count and background (arbitrary units). With 5 mM Mg²⁺, SSB increases DNAp binding (N = 5–8, p < 0.05). Data presented as mean ± SEM. Statistical comparisons were performed using Welch’s unpaired two-tailed t-tests. Source data are provided as a Source Data file.