Fig. 4: Essentiality of SSB saturation and C-terminal tail for functional replication dynamics.

A Two-dimensional free energy profile of T7 SSB-Δ21C (C-terminal truncated mutant) binding to ssDNA in the presence of DNAp-trx complex. Energy barrier between states increased from ~3 kcal/mol (wild-type) to ~10 kcal/mol, hindering state transitions and demonstrating the C-terminal tail’s role in facilitating displacement. B Detailed view of C-terminal SSB binding to DNAp during MD simulation. Orange: coarse-grained ssDNA segments; red: SSB C-terminal tail; green: DNAp front basic patch region. Key positive residues (K587, K589, R590, R591, K594, and R599) colored blue. Critical electrostatic interactions shown: R590-E214 distance (5.6 Å) and R599-E232 distance (8.4 Å), demonstrating strong binding between the basic patch and acidic C-terminal. C Distance variation between DNA and SSB during unbiased simulations comparing DNAp+gp2.5 (blue) versus DNAp+gp2.5-Δ21C (orange). Full-length gp2.5 shows progressive detachment from ssDNA over simulation time, contrasting with persistent binding of the Δ21C mutant, confirming the C-terminal’s role in displacement facilitation. D, E Real-time primer extension assays with escalating wild-type (D) and mutant (E) SSB concentrations (0–3000 nM). Black arrows indicate minimum fluorescence intensity marking transition from polymerase-dominated to exonuclease-dominated phases. N = 3 replicates per concentration. F Quantified polymerase activity as a function of varying SSB concentrations. Activity estimated from initial linear curve section (~1.5 min) representing polymerase-dominated phase. Wild-type SSB increases replication rate (~20% enhancement from 0 to 3000 nM), while mutant SSB decreases the rate (~25% reduction), with effects apparent only above 150 nM. N = 3 replicates per concentration. G, J Single-molecule force spectroscopy analysis of replication rates at 10 pN (G) and 20 pN (J) with varying SSB concentrations (10, 30, 150 nM). Dashed lines: reference rates without SSB. H, K Exonuclease binding probability analysis at 10 pN (H) and 20 pN (K) forces. Probability calculated as the ratio of exonuclease events to total polymerase plus exonuclease events per DNA molecule. Wild-type SSB promotes polymerase mode; mutant favors exonuclease mode. I, L Average pause duration comparison at 10 pN (I) and 20 pN (L) forces with varying SSB concentrations. Wild-type SSB increases pausing at low forces due to recruitment effects. Mutant SSB increases pausing at all concentrations, demonstrating roadblock formation without productive displacement. Data presented as mean ± SEM. Source data are provided as a Source Data file.