Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2

Abstract

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2.

Fig. 1. RPA specifically promotes both helicase and nuclease activities of Dna2.

a A schematic of RPA. b Purified wild-type RPA used in this study. c Recombinant S. cerevisiae Dna2 and Dna2-E675A (nuclease-dead) variants used in this study. d Yeast RPA and human mitochondrial SSB (mtSSB) were used in nuclease assays with Dna2 and 5′- overhanged DNA substrate (45 nt ssDNA, 48 bp dsDNA, 1 nM, in molecules). The red asterisk indicates the position of the radioactive label. e, f Quantification of nuclease assays such as shown in panel d. Error bars, SEM; n = 3. g Yeast RPA and mtSSB were used in helicase assays with Dna2-E675A and 5′-overhanged DNA substrate (30 nt ssDNA, 31 bp dsDNA, 1 nM, in molecules) with 50 mM KCl. h Quantification of assays such as shown in panel g. Error bars, SEM; n = 4.

Fig. 2. RPA stimulates Dna2 beyond recruitment to DNA.

a Representative electrophoretic mobility shift assays to monitor recruitment of Dna2-E675A to either free or RPA/mtSSB-precoated (8 nM) 5′-overhanged DNA substrate (45 nt ssDNA, 48 bp dsDNA, 1 nM, in molecules) and with 150 mM NaCl. The red asterisk indicates the position of the radioactive label. b Quantification of assays such as shown in panel a. Error bars, SEM; n = 3. c A scheme of molecular weight determination using mass photometry. Scattered light from single adsorbed biomolecular complexes interferes with light reflected at the surface. The detected single-molecule interference contrast is proportional to the molecular weight. d Measured molecular weight distributions of RPA and Dna2 complexes in absence and presence of a 5′-overhanged DNA substrate (25 nt ssDNA, 48 bp dsDNA, 20 nM, in molecules). For the formation of a heterotrimeric complex (panel at the bottom), RPA was added first to the DNA followed by Dna2 addition. e DNA binding of Dna2-E675A to forked DNA (45 nt ssDNA, 48 bp dsDNA, 0.1 nM, in molecules) and with 50 mM KCl. One out of two independent experiments is shown. f Unwinding of forked DNA as in panel e by Dna2-E675A. DNA substrate was first pre-bound by saturating concentrations of Dna2-E675A (25 nM) before adding yeast RPA or human mtSSB (both 48 nM) together with ATP and with 50 mM KCl. One out of three independent experiments is shown.

Fig. 3. The DBD-FAB domains of Rfa1 stimulate the Dna2 nuclease.

a A scheme of wild type RPA and selected point mutants. K_D, concentration of the respective RPA variant resulting in 50% binding of ssDNA (93 nt, 0.1 nM, in molecules) such as shown in Supplementary Fig. 3c, h. Error, SEM; n = 3. The extent of stimulation of the Dna2 nuclease is indicated on the right. b Quantitation of ssDNA (93 nt, 0.1 nM, in molecules) binding by the RPA variants as shown in panel a. Error bars, SEM; n = 3. c Quantification of nuclease assays with his-tagged RPA wild type and point mutants and Dna2 using 5′-overhanged DNA (45 nt ssDNA, 48 bp dsDNA, 1 nM in molecules). Error bars, SEM; n = 3. d A scheme of RPA subunits and Rfa1 fragments. K_D, concentration of the respective variant resulting in 50% binding to ssDNA (93 nt, 0.1 nM, in molecules), such as shown in Supplementary Fig. 3k, l; Error, SEM; n = 3. The extent of stimulation of the Dna2 nuclease is indicated on the right, based on nuclease assays such as shown in panels f and i. e Quantitation of ssDNA (93 nt, 0.1 nM, in molecules) binding by Rfa1 fragments shown in panel d. Error bars, SEM; n = 3. f Representative nuclease assays showing degradation of 5′-overhanged DNA (45 nt ssDNA, 48 bp dsDNA, 1 nM in molecules) by Dna2 and its stimulation by the RPA subunits. The red asterisk indicates the position of the radioactive label. g Quantification of nuclease assays such as shown in panel f. RPA wild type is replotted as in panel c for reference. Error bars, SEM; n = 3. h Representative nuclease assays as in panel f, but with shorter 5′-overhanged DNA (19 nt ssDNA, 31 bp dsDNA, 1 nM, in molecules) and with 100 mM NaCl. i Representative nuclease assays with Rfa1 fragments and Dna2 using 5′-overhanged DNA (45 nt ssDNA, 45 bp dsDNA, 1 nM, in molecules). j Quantification of nuclease assays such as shown in panel i. Rfa1 is replotted as in panel g for reference. Error bars, SEM; n = 3. k Quantification of nuclease assays such as shown in Supplementary Fig. 3m, n. Error bars, SEM; n = 3.

Fig. 4. Dna2 recruitment and nuclease stimulation by RPA are genetically separable.

a A schematic representation of the domain organization in Dna2 and RPA. b A scheme of the interaction between the N-terminal domain of S. cerevisiae Rfa1 (DBD-F) and the predicted helix within the N-terminal part of Dna2 generated by comparative modeling using the structure of the orthologous human complex (PDB: 5EAY). Residues I14 and F11 mutated in this study are highlighted. c Multiple sequence alignment of RPA1/Rfa1 orthologs from various species focused on the N terminal region of RPA1, emphasizing the conserved positions located in the interface of the structural model of the complex between yeast Rfa1 and Dna2. d A schematic representation of the primary structures of RPA-SSL mutant. Wild type RPA and RPA-I14S are again shown as reference. K_D, concentration of the respective RPA variant resulting in 50% binding to ssDNA (93 nt, 0.1 nM, in molecules,) such as shown in Supplementary Fig. 4b; Error, SEM; n = 3. e Quantitation of ssDNA (93 nt, 0.1 nM, in molecules) binding by wild type RPA and RPA-SSL mutant in assays such as shown in Supplementary Figs. 3c and 4b. RPA is replotted as in Fig. 3b for reference. Error bars, SEM; n = 3. f Representative nuclease assays using Dna2 and 5′-overhanged DNA substrate (45 nt ssDNA, 48 bp dsDNA, 1 nM, in nucleotides) performed as in Fig. 1d, but with 100 mM NaCl. The red asterisk indicates the position of the radioactive label. g Quantification of nuclease assays such as shown in panel f. Error bars, SEM; n = 3. h Quantification of DNA binding assays such as shown in Supplementary Fig. 4g. Wild type Dna2 was bound to 5′-overhanged DNA substrate (45 nt ssDNA, 48 bp dsDNA, 1 nM, in molecules) pre-coated with either wild type RPA or RPA-SSL in the presence of 3 mM EDTA and 150 mM NaCl. Bars indicate range; n = 2. i Quantification of experiments as in panel h, but with Dna2-E675A in the presence of 5 mM Mg²⁺ and 150 mM NaCl. RPA is replotted as in Fig. 2a for reference. Bars indicate range; n = 2. j Quantification of assays such as shown in Supplementary Fig. 4h showing Dna2-E675A binding to 5′-overhanged DNA substrate (30 nt ssDNA, 31 bp dsDNA, 1 nM, in molecules) pre-coated with RPA, Rfa1-FAB, Rfa1-ABC or Rfa1-AB fragments in the presence of 5 mM Mg²⁺ and 150 mM NaCl. Error bars, SEM; n = 3. k Quantification of assays such as shown Supplementary Fig. 4i. The respective RPA variant was tested for capacity to protect 3′-overhanged DNA substrate (45 nt ssDNA, 48 bp dsDNA, 1 nM, in molecules) from Dna2 degradation with 100 mM NaCl. Error bars, SEM; n = 4.

Fig. 5. RPA-I14S and RPA-SSL mutants promote the Dna2-E675A helicase.

a Unwinding of a Y-shaped DNA substrate (45 nt ssDNA, 48 bp dsDNA 0.1 nM, in molecules) by Dna2-E675A in the presence of RPA variants and 50 mM KCl. The red asterisk indicates the position of the radioactive label. b Quantification of assays such as shown in panel a. Error bars, SEM; n = 3. c Quantification of 2.2-kbp-long dsDNA substrate (0.1 nM, in molecules) unwinding by Dna2-E675A in the presence of RPA variants. Error bars, SEM; n = 3. d A sketch of the employed magnetic tweezers assay including the DNA construct carrying a 40-nt-long 5′-ssDNA flap to allow loading of Dna2-E675A (top). DNA unwinding trajectory with sequential addition of reagents revealing that DNA unwinding was only observed after the addition of Dna2-E675A. e Representative trajectories of DNA unwinding events by Dna2-E675A (5 nM). The reaction was supplemented with RPA (20 nM). f, g Velocity histogram and cumulative probability distribution (shown as survival probability) of the processivity obtained from DNA unwinding trajectories of Dna2-E675A in presence of RPA. Error, SEM; n = 25. h Representative trajectories of DNA unwinding by Dna2-E675A (5 nM). The reaction was supplemented with RPA-I14S (20 nM). i, j Velocity histogram and cumulative probability distribution (shown as survival probability) of the processivity obtained from DNA unwinding trajectories of Dna2-E675A in presence of RPA-I14S. Error, SEM; n = 29. k Quantification of 2.2-kbp-long dsDNA substrate (0.1 nM, in molecules) unwinding by Dna2-E675A in the presence of RPA, subunits of RPA and fragments of Rfa1, as indicated. Error bars, SEM; n = 3. l Quantification of Y-shaped DNA substrate (45 nt ssDNA, 48 bp dsDNA 0.1 nM, in molecules) unwinding such as shown in Supplementary Fig. 5c. RPA is replotted as in panel b for reference. Error bars, SEM; n = 3. m Bar graph of processivities of DNA unwinding events by Dna2-E675A (5 nM) in the presence of RPA variants (20 nM). The distribution of the data is shown in Fig. 5g, j and Supplementary Fig. 5f, i. Error, SEM; n = 25, 25, 30 and 22 for RPA, Rfa1, Rfa1-FAB and Rfa1-ABC. Representative traces are shown in Supplementary Fig. 5d, g. n Bar graph of velocities of DNA unwinding by Dna2-E675A (5 nM) in the presence of RPA variants (20 nM). The distribution of the data is shown in Fig. 5f, i and Supplementary Fig. 5e, h. Error, SEM; n = 25, 25, 30 and 22 for RPA, Rfa1, Rfa1-FAB and Rfa1-ABC. Representative traces are shown in Supplementary Fig. 5d, g.

Fig. 6. The DBD-A and DBD-B domains of Rfa1 specifically promote the Dna2 helicase.

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. K_D, 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. K_D, 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.

Fig. 7. RPA-SSL and SKYD mutants are impaired in DNA end resection.

a A representative experiment to monitor DNA end resection by Sgs1, Dna2, and increasing concentrations of yeast RPA variants using 2.2-knt-long dsDNA (0.5 nM, in molecules). The reaction buffer contained 100 mM NaCl. Red asterisks indicate random radioactive labels on the DNA. b Quantification of overall substrate utilization from experiments such as shown in panel a. Error bars, SEM; n = 3. c A model showing that RPA first recruits Dna2 to 5′-overhanged DNA (1). Subsequently, RPA remains a component of the nucleoprotein complex and stimulates both nuclease (Nuc) and ATPase-driven translocase (Hel) activities of Dna2 (2). d A model depicting domains of the RPA large subunit, Rfa1, and their involvement in ssDNA binding, recruitment of Dna2 and stimulation of nuclease and helicase activities of Dna2, respectively.