Fig. 6

Biophysical assessment of an Rfa1 phosphomimetic mutant. a Microscale thermophoresis (MST) binding curves of DBD-A^S178D mutant versus Rfa3-OB. b, c MST-binding curves of RPA heterotrimeric complexes interacting with ssDNA, using wild-type RPA (red, RPA^wt) and RPA phosphomimetic mutant S178D (blue, RPA^S178D) and a Cy5-labeled dT₁₀₀ ssDNA substrate. For all MST curves, apparent EC₅₀ values were calculated using the Hill-equation. Normalized fluorescence was calculated using NanoTemper Analysis 1.2.101. Measurements were repeated five times and data points are averages shown with SEM, maximum fluorescence (Bmax), and R². d The Hill coefficients for the RPA-dT100-binding studies were calculated from the hill-plot transform of the binding data. e Schematic of the FRET-based experiment using fluorescent versions of RPA. f Based on the relationship between DBD-A and Tri-C (Fig. 3c–f) we estimated a distance of 45 Å between the fluorescent label sites. g, h Stopped flow fluorescence measurements of g RPA^WT-DBD-D-Cy5 and RPA^WT-DBD-A-Cy3 and h RPA^S178D-DBD-D-Cy5 and RPA^S178D-DBD-A-Cy3 FRET complex formation on different lengths of ssDNA (dT₂₀–dT₉₇). No ssDNA controls are shown in gray (denoted dT₀). FRET signal is only produced only when ssDNA ≥ 45nt is present. i, j Stopped flow experiments and analysis of i RPA^WT-DBD-D-Cy5 and RPA^WT-DBD-A-Cy3 and j RPA^S178D-DBD-D-Cy5 and RPA^S178D-DBD-A-Cy3 as a function of increasing length of ssDNA (nucleotides, n). Data were fitted with a two-step model and the observed rate of association (RPA–ssDNA or RPA–RPA association) plotted against nucleotide number. i The rate of association (k_obs,1) for RPA^WT remains consistent with increasing ssDNA length. j An increase in rate of association (k_obs,1) correlated with an increase in ssDNA length is observed for the RPA^S178D variant and suggests a cooperative-binding behavior of this mutant. Data points are an average of three experiments and are shown with SEM. Source data are provided as a Source Data file