Fig. 6: The DBD-A and DBD-B domains of Rfa1 specifically promote the Dna2 helicase.
From: Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2
a A schematic representation of the domain organization in Dna2 and RPA, highlighting with a dashed gray box the domains used as inputs of the free docking simulation. Right, most likely structural model obtained after free docking with the InterEvDock2 server. Dna2 nuclease-helicase domain and the AB module of Rfa1 are represented in pink and green, respectively, ssDNA is in dark gray. Lower left panel focuses on the interaction region of Rfa1 (in green) with Dna2 (in pink), highlighting the conserved residues S191 and Y193 of Rfa1. Y193 is predicted to anchor in an apolar pocket exposed at the surface of Dna2 formed by V517, V684, V688 and I731. Bottom, pairwise sequence alignment between S. cerevisiae Rfa1 and human RPA1 sequences in the region 185–200 highlights the conservation of the Y193 residue. b A schematic representation of the primary structure of wild type Rfa1-AB and point mutants. Rfa1-AB is again shown as reference. KD, concentration of the respective Rfa1-AB variant resulting in 50% binding to ssDNA (93 nt, 0.1 nM, in molecules) such as shown in Supplementary Fig. 6c. Error, range; n = 2. c Quantitation of Rfa1-AB variants binding to ssDNA (93 nt, 0.1 nM, in molecules) as shown in Supplementary Fig. 6c. Rfa1-AB is replotted as in Fig. 3e for reference. Bars show range; n = 2. d Representative experiments showing unwinding of 2.2-kbp-long dsDNA (0.1 nM, in molecules) by Dna2-E675A in the presence of Rfa1-AB variants. Red asterisks indicate random radioactive labels on the DNA. The experiment was performed three times with similar results. e Primary structure of the RPA-SKYD mutant. Wild type RPA is again shown as a reference. KD, concentration of the respective Rfa1-AB variant resulting in 50% binding to ssDNA (0.1 nM, in molecules, 93-nt-long) such as shown in Supplementary Fig. 6f. Error, SEM; n = 3. f DNA binding by RPA-SKYD to ssDNA (93 nt, 0.1 nM, in molecules) as shown in Supplementary Figs. 3c and 6f. RPA is replotted as in Fig. 3b for reference. Error bars, SEM; n = 3. g Representative experiments showing unwinding of 2.2-kbp-long dsDNA (0.1 nM, in molecules) by Dna2-E675A in the presence of wild type RPA or RPA-SKYD. h Quantification of helicase assays such as shown in panel g. Error bars, SEM; n = 3. i Apparent ATP turnover number and its dependence on various RPA variants (48 nM) in the presence of single-stranded DNA (50 nt, 1 μM, in nucleotides). The reactions contained Dna2 E675A (1 nM) and ATP (1 mM). Error bars, SEM, n = 3. j Quantification of nuclease assays such as shown in Supplementary Fig. 6g showing kinetics of degradation of 2.2-knt-long ssDNA (0.3 nM) by Dna2 in the presence of RPA variants (50 nM) and with 100 mM KCl. Error bars, SEM; n = 3.
