Fig. 3: The DBD-FAB domains of Rfa1 stimulate the Dna2 nuclease.
From: Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2
a A scheme of wild type RPA and selected point mutants. KD, 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. KD, 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.
