Fig. 5: Tyrosine 193 in Rfa1 is critical for processive ssDNA digestion by the Dna2–RPA ensemble.

a Digestion by Dna2 on 5′-labeled 40-nt 5′-overhanging ssDNA (5 nM) with Rfa1–NAB and Rfa1–NAB aromatic mutants. Percentage of digestion from one-time screening experiments was listed with partially defective mutants underlined. b Digestion by Dna2 on 5′-labeled 40-nt 5′-overhanging ssDNA (5 nM) with Rfa1–NAB and rfa1–NAB–Y193A mutant. For quantification, mean values ± s.d. from three independent experiments were plotted. c Digestion by Dna2 on 3′-labeled 40-nt 5′-overhanging ssDNA (5 nM) with Rfa1–NAB and rfa1–NAB–Y193A mutant. The experiments were repeated three times. d Wide-field image of a Dna2 (magenta punctum)–RPA–Y193A–GFP complexes digesting the ssDNA–RPA–Y193A–GFP complexes (green) (Exp. 7 in Supplementary Table 1). e Processivity distribution of single Dna2 digestion with RPA–Y193A in solution (Exp. 7 in Supplementary Table 1): 130-nm bin. Digestion events N = 18. Each experimental condition was examined over more than three DNA Curtains experiments (n ≥ 3). Error bars were obtained through the bootstrap analysis. For any normally distributed dataset, 68.27% of the values lie within one standard deviation of the mean, therefore, our choice of 70% confidence intervals for the bootstrapped data provides a close approximation to expectations for one standard deviation from the mean. The data were fitted with Gaussian functions (red dash line). The errors represented 95% confidence intervals obtained through Gaussian function fitting. f Dna2 recruitment (%) with RPA–Y193A in solution (Exp. 7 in Supplementary Table 1). Independent DNA Curtains experiments were repeated: n = 3 for Exp. 2; n = 3 for Exp. 7; Error bars, mean ± s.d. Statistical significance was analyzed using the unpaired t-test for two groups. p-value: two-tailed; p-value style: GP: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). Confidence level: 95%. g Digestion by Dna2 on 5′-labeled 40-nt 5′-overhanging ssDNA (5 nM) with Rfa1–NAB and rfa1–NAB–K45E mutant. For quantification, mean values ± s.d. from three independent experiments were plotted. h Formation of ternary complex from dna2–D657A, Rfa1–NAB variants, and dT₍₂₀₎ ssDNA (5 nM). The experiments were repeated three times. Source data are provided as a Source Data file.