All RPA mutant heterotrimeric proteins exhibit increased affinity for ssDNA, and RPA^V227A and RPA^E240K possess increased capacity to unfold human telomeric G-quadruplex (h-telG4) DNA. (A) Schematic depiction of the experimental schemes for the Förster resonance energy transfer (FRET)-based assays. Binding of RPA:RPA1^WT (RPA^WT), RPA:RPA1^V227A (RPA^V227A), RPA:RPA1^V270A (RPA^V270A), and RPA:RPA1^E240K (RPA^E240K) proteins was monitored by using 1 nM dT30 ssDNA molecules (top), 1 nM dT15, or TTAGGGTAAGGGTAA telomeric DNA sequence (middle) labeled with Cy3 and Cy5 fluorescent dyes at the 5′ and 3′ ends, respectively. High FRET corresponds to free ssDNA, and low FRET reflects RPA binding. Unfolding of the h-telG4 was monitored by using the (TTAGGG)₅ sequence (bottom). FRET between the Cy3 and Cy5 dyes calculated for the h-telG4 in the absence of proteins and in buffer containing K⁺ corresponds to 100% folded quadruplex, whereas the FRET value of h-telG4 in the presence of saturating concentrations of RPA^E240K in buffer containing Li⁺ corresponds to 100% unfolded h-telG4. (B) Stoichiometric binding (1 RPA: 1 dT30 molecule) was observed for RPA^E240K, and nearly stoichiometric binding was observed for RPA^WT, RPA^V227A, and RPA^V270A. The arrows mark the respective protein concentrations at inflection points of the 2-line linear regression fit. dT15 (C) and TTAGGGTAAGGGTAA (D) telomeric DNA sequence binding to RPA^WT, RPA^V227A, RPA^V270A, and RPA^E240K. The data were fitted to a quadratic binding equation. The calculated K_ds with respective fitting errors are listed in supplemental Table 3. (E-F) Melting of the h-telG4 DNA, stabilized by the presence of 100 mM KCl. (E) Extent of the h-telG4 melting reactions was calculated from the plateaus of each respective time course. (F) h-telG4 melting rates for RPA^WT, RPA^V227A, RPA^V270A, and RPA^E240K were calculated from the slopes of FRET change during the first 20 seconds of each time course (supplemental Figure 4). The data were fitted to a quadratic binding equation. The calculated apparent K_ds with respective fitting errors are listed in supplemental Table 3. In all panels, the data are shown as average for 3 independent experiments. Error bars represent standard deviation. Where not shown, error bars are smaller than the data points.