Fig. 2: Single-molecule visualization of T7 DNAp displacing T7 SSB.

A Experimental design combining dual-optical trapping with dual-color imaging to investigate SSB displacement mechanisms during replication. Saturated SSB (150 nM, 4% labeled) tests two displacement models: sequential one-by-one SSB removal versus a collective whole-train pushing mechanism. B Representative mechanical trace illustrating DNAp-SSB interactions with force transition from 45 to 10 pN (dashed line). Junction position derived from independent optical tweezer measurements exploiting elasticity differences between ssDNA and dsDNA, enabling real-time DNAp tracking during replication. C Concurrent confocal kymographs capturing dynamic interactions between fluorescently labeled DNAp (top) and SSB (bottom) for the molecule shown in (B). Yellow line: force transition. Scale: 10 s, 75 nm/pixel. D Composite analysis combining mechanical data from (B) with fluorescence data from (C) to directly observe DNAp-SSB interactions. Only replication processes are displayed for clarity. Fluorescently labeled SSB visualized binding to ssDNA via fluorescence microscopy. Only SSB trajectories >3 pixels were analyzed for reliability. Total analysis: 58 DNAp-SSB interaction traces extracted from 25 unique DNA molecules (from 64 total molecules) exhibiting clear, consistent fluorescence signals. E Three representative distance dynamics between DNA polymerase and SSB during replication. Top panel based on data from (D). Green: processive replication; blue: backtracking; gray: pausing. Time axes rescaled for presentation clarity. F Histogram showing distance changes during displacement events. Analysis limited to first burst segments accounting for potential bidirectional DNAp movement. Negative values indicate backtracking events. Of 58 analyzed events: 15 show DNAp retreat due to backtracking (light blue), 36 indicate DNAp approach through SSB displacement (light green), and seven remain stationary, likely due to pausing. Results strongly support the one-by-one SSB removal model. G T7 SSB diffusion constant calculated using the mean square displacement method reveals predominantly stationary behavior on ssDNA (7 ± 7 × 10⁻⁴ μm²/s). Approximately 10% of measurements showing negative values were excluded as noise artifacts. H Example traces demonstrating displacement of fluorescently labeled SSB by DNAp under 10 pN force. Green line represents the DNAp trajectory. Source data are provided as a Source Data file.