Fig. 3: A phosphomimetic S384D substitution minimally influences the DNA binding properties of RPA but induces configurational changes.

a Contacts between Arg-384 and the ssDNA are shown in the crystal structure of DBD-A, B (PDB:1JMC). Ser-384 does not directly contact the ssDNA, but positions Phe-386 to promote a key base stacking interaction with the base. RPA or RPA^S384D binding to DNA was measured by fluorescence anisotropy using either (b) 5′-FAM-(dT)₂₀ or (c) 5′-FAM-(dT)₄₀ ssDNA oligonucleotides. In both cases, high-affinity stoichiometric binding is observed for RPA and RPA^S384D and no significant changes in the binding behavior are observed. Stopped flow kinetic analysis of ssDNA binding was performed with (d) RPA or (e) RPA^S384D by monitoring the change in intrinsic tryptophan fluorescence as a function of ssDNA (dT)₃₅ concentration. f Plot of the k_obs versus ssDNA concentration yields k_on and k_off values. RPA^S384D has a slower k_on (1.9 ± 0.1 × 10¹⁰ M⁻¹ s⁻¹) and faster k_off (65 ± 7 s⁻¹) compared to RPA^WT (k_on = 5.2 ± 0.3 × 10¹⁰ M⁻¹ s⁻¹ and k_off = 39 ± 4 s⁻¹). K_D values extracted from these measurements show RPA^WT binding to ssDNA with ~5-fold higher affinity (K_D = 0.75 ± 0.4 nM) compared to RPA^S384D (K_D = 3.4 ± 0.2 nM). g A Förster Resonance Energy Transfer (FRET) experiment was developed using two fluorescent versions of RPA. RPA was site-specifically labeled on either DBD-A with Cy3 or DBD-D with Cy5. Equimolar ratios of both fluorescent RPA were mixed with ssDNA (dT)₉₇ in a stopped flow. Changes in Cy5 fluorescence were monitored by exciting Cy3 at 535 nm. Assembly of multiple RPA on the long ssDNA substrate results in a high FRET signal (green trace). In the absence of ssDNA, no enhancement in fluorescence is observed. h RPA filaments were pre-formed on ssDNA (dT)₉₇ using the Cy5- and Cy3-labeled RPA and facilitated exchange activity was measured by challenging the RPA^Cy3-RPA^Cy5-ssDNA assembly with mixing against unlabeled RPA or RPA^S384D. RPA exchanges the fluorescent-RPA faster (k_FE = 1.8 s⁻¹) compared to RPA^S384D (k_FE = 0.9 s⁻¹). Data are presented as ±SEM from three independent experiments.