Fig. 3: Active displacement of SSB by DNA polymerase through direct physical interaction.
From: DNA polymerase actively and sequentially displaces single-stranded DNA-binding proteins
A Structural details of the predicted full-length SSB monomer using AlphaFold2 and Modeller. Gray: template 1JE5 regions; orange: positive binding groove; red: AlphaFold2 C-terminal; blue: Modeller C-terminal. B Free energy profile for SSB binding to ssDNA process using umbrella sampling simulations. Profile reveals three distinct local minima characterizing SSB-ssDNA binding: bound state (1.3–2.0 nm), intermediate state (~2.7 nm), and dissociated state (3.5–4.0 nm). C Representative structure of intermediate state from (B). ssDNA (blue) and C-terminal tail (red) positioned at opposite sides of positive binding groove, indicating competitive binding scenario between C-terminal and ssDNA for groove occupancy, stabilizing intermediate state. D Two-dimensional free energy profile of SSB-ssDNA binding incorporating electrostatic interactions between the positive binding groove and ssDNA as an additional coordinate. Representative structures from each local energy basin are shown. E Structural details of DNAp-thioredoxin-SSB complex bound to DNA using AlphaFold2-predicted SSB positioned based on 6P7E template. Green: full-length DNAp structure; blue: thioredoxin; gray/orange/red: SSB majority/positive binding groove/C-terminal tail, respectively. dsDNA with 5’ ssDNA overhang displayed. F Two-dimensional free energy profile of SSB-ssDNA binding in the presence of DNAp-trx complex. Bound state becomes relatively unstable compared to SSB alone, with the system trapped in a dissociated state. High electrostatic effects from SSB C-terminal promote dissociation. G Top: Schematic explaining FRET proximity measurement setup. Bottom: Representative replication activity from mechanical measurements showing base pair changes over time. Regions with constant base pairs (pausing) and reductions (backtracking) correlate with FRET signal regions (light blue shaded areas from panel H). H Representative kymograph of DNAp-SSB interaction. Top: SSB fluorescence; middle: DNAp fluorescence (green excitation only); bottom: merged image. Yellow line: real-time DNAp position from mechanical measurements. Images processed to reduce background noise, correct signal crosstalk, and filtered for clarity. I Analysis of eight individual FRET events selected from 16 DNA molecules based on fluorescence signal quality. Corresponding base pair-time traces extracted with FRET signal regions highlighted (light blue). FRET signals were detected during pausing events (6/8 cases) and backtracking events (2/8 cases), not observed during active replication, supporting the direct physical interaction model.
