Figure 1.

Visualization of Rep binding to ssDNA. (A) Schematic representation of Rep proteins binding to dT₃₅ oligos in surface plasmon resonance investigations. (B–C) SPR sensorgrams of (B) 20 nM Rep WT association (60 s) and dissociation from dT₃₅ (dark gray), dT₁₅ (gray) and dT₁₀ (light gray). Sensorgrams for Rep binding to dT₁₀ and dT₁₅ were normalized to that for dT₃₅ to enable comparison of binding to the same molar surface density of immobilized oligos. Based on the responses and molecular weights of Rep and oligos, it is estimated that ∼1050 RU of bound Rep corresponds to a stoichiometry n= 1.0 Rep monomers bound to each oligo (Equation 1). (C) Response of dT₃₅-bound Rep WT to interrogation by 200 μM AMP-PNP (I), ADP (II), and ATP (III) injection at 20 μL min^–1 for 60 s. Only ATP resulted in fast dissociation of Rep from dT₃₅. (D) Schematic representation of the single-molecule Rep-AF647-DNA binding assay. Rolling-circle DNA templates (2030 bp) are pre-incubated with DnaBC and applied to a microfluidic flow cell. The 5′-biotinylated DNA template couples to the streptavidin functionalized coverslip. Rep-AF647, in the presence of SSB, ATP, and magnesium, is then applied to the flow cell and imaged to monitor for binding. (E) Example kymographs of Rep-AF647 (magenta – bottom) binding to DNA templates (gray – top) in the presence and absence of 5 mM ATP. Arrows indicate the time point of Rep-AF647 addition to the flow cell. (F) Heatmap of the number of Rep-AF647 monomers bound to the DNA template over time in the presence of ATP (n = 65).