Abstract
During homologous recombination, Rad51 forms a nucleoprotein filament with single-stranded DNA (ssDNA) that undergoes strand exchange with homologous double-stranded DNA (dsDNA). Here, we use real-time analysis to show that strand exchange by fission yeast Rad51 proceeds via two distinct three-strand intermediates, C1 and C2. Both intermediates contain Rad51, but whereas the donor duplex remains intact in C1, the ssDNA strand is intertwined with the complementary strand of the donor duplex in C2. Swi5–Sfr1, an evolutionarily conserved recombination activator, facilitates the C1–C2 transition and subsequent ssDNA release from C2 to complete strand exchange in an ATP-hydrolysis-dependent manner. In contrast, Ca2+, which activates the Rad51 filament by curbing ATP hydrolysis, facilitates the C1–C2 transition but does not promote strand exchange. These results reveal that Swi5–Sfr1 and Ca2+ have different activation modes in the late synaptic phase, despite their common function in stabilizing the presynaptic filament.
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Acknowledgements
We thank Y. Kokabu, M. Ikeguchi, L. Reha-Krantz, H. Taguchi, J. E. Haber and F. Uhlmann for discussions. We express special thanks to B. Argunhan for critical reading of the manuscript. This study was supported partly by Grants-in-Aid for Scientific Research on Innovative Areas (15H059749 to H.I.) and for Young Scientists (A) (16H06160 to Y.M.) and a Research Fellowship (15J08408 to K.I.) from the Japan Society for the Promotion of Science (JSPS).
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K.I., Y.M., M.T., and H.I. conceived the study and designed the research. K.I. performed all experiments. K.I., Y.M., M.T., and H.I. analyzed the data. K.I. and H.I. wrote the manuscript.
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Supplementary Figure 1 FRET-based assays to monitor Rad51-mediated DNA-strand exchange in real time
a Fluorescence anisotropy showing that Rad51 filaments are stable for at least 60 min. The reactions contained Rad51 (1.5 µM) or Rad51 (1.5 µM) + Swi5-Sfr1 (0.15 µM) in buffer A (30 mM HEPES-KOH [pH 7.5], 1 mM DTT, 0.1% [w/v] BSA, and 0.0075% [w/w] Tween-20) containing 0.25 mM ATP and 15 mM MgCl2 and were performed at 37°C. b Residuals between experimental data of the DNA strand pairing assay and a theoretical curve obtained by simulation. (a) Three-step model. (b) Two-step model. (c) Merged (a) on (b). The standard pairing reaction was carried out (Mg2+ + ATP). Note that the x-axis shows the logarithm of reaction time (s). Residuals represent the variation between experimental data and the theoretical curves of the indicated model. The residuals for the experimental data and the two-step model present a systematic deviation that gave a negative bias at a very early stage (within ~20 seconds) and a positive bias at a second early stage (within ~20–150 seconds). In contrast, the residuals for the experimental data and the three-step model present a random deviation. The F values were calculated using the two residuals (A and B) from the initial 100 secs. An F value larger than F 0.01 (= 1.60) indicates that the variance of residuals is significantly different (P < 0.01). The three-step model showed a better fit than the two-step model, especially at early time points.
Supplementary Figure 2 Residuals between experimental data of DNA-strand-pairing assays in Figs. 2 and 3 and a theoretical curve obtained by simulation using the two-step or three-step model
Blue and red lines show the residuals from simulation of the two-step and three-step models, respectively. The F values were calculated using the two residuals (two-step and three-step) from the initial 100 secs. An F value larger than F 0.01 (= 1.60) indicates that the variance of residuals is significantly different (P < 0.01). Under each reaction condition, the three-step model shows a better fit than the two-step model.
Supplementary Figure 3 Impact of substrate concentrations on the DNA-pairing assay
a Time course of strand exchange in the pairing assay with various concentrations of Rad51-ssDNA filament and dsDNA. In this assay, the ratio of Rad51-ssDNA filament and dsDNA was kept constant (Rad51-ssDNA filament : dsDNA=1:1) b Time course of the standard pairing assays (36 nM each of the substrates). The concentration of Swi5-Sfr1 was 0.15 µM, which was one-tenth of the Rad51 concentration. c Time course of the pairing assays containing 72 nM substrates. The concentration of Swi5-Sfr1 was 0.3 µM, which was one-tenth of the Rad51 concentration. d Reaction rate constants [k1 (a), k−1 (b), k2 (c), k−2 (d), k3 (e) and k−3 (f)] of each reaction in Fig. S4A, B and C were calculated by simulation using the three-step model. The error bars represent ± s.d.(n = 3). e Residuals between experimental data of DNA strand pairing assays and a theoretical curve obtained by simulation using the two-step or three-step model. Blue and red lines show the residuals from simulation of the two-step and three-step models, respectively. The F values were calculated using the two residuals (two-step and three-step) from the initial 100 secs. An F value larger than F 0.01 (= 1.60) indicates that the variance of residuals is significantly different (P < 0.01). Under each reaction condition, the three-step model shows a much better fit than the two-step model.
Supplementary Figure 4 Analysis of Rad51-driven DNA strand exchange stimulated by Ca2+
a Residuals between experimental data of DNA strand pairing assays in Fig. 4 and a theoretical curve obtained by simulation using the two-step or three-step model. Blue and red lines show the residuals from simulation of the two-step and three-step models, respectively. The F values were calculated using the two residuals (two-step and three-step) from the initial 100 secs. An F value larger than F 0.01 (= 1.60) indicates that the variance of residuals is significantly different (P < 0.01). Under each reaction condition, the three-step model shows a better fit than the two-step model. b Ca2+ renders DNA strand pairing reactions refractory to the stimulatory effects of Swi5-Sfr1. DNA strand pairing assays were conducted under the same conditions as in Fig. 4A (1.5 µM Rad51 and 0.15 µM Swi5-Sfr1).
Supplementary Figure 5 Rad51 does not affect the fluorescence emission of fluorescein or the quenching efficiency of rhodamine
a Fluorescence spectra of the pairing assay. Curve colors are as follows: blue, 83 mer ssDNA labeled with fluorescein at the 5’ end; red, 83 mer ssDNA labeled with fluorescein at the 5’ end that has been bound by Rad51 to form the presynaptic filament; green, dsDNA consisting of 83 mer ssDNA labeled with fluorescein at the 5’ end and 40 mer ssDNA labeled with rhodamine at the 3’ end; purple, complex of Rad51 and dsDNA consisting of 83 mer ssDNA labeled with fluorescein at the 5’ end and 40 mer ssDNA labeled with rhodamine. b Fluorescence spectra for the displacement assay. Curve colors are as follows: blue, 40 mer ssDNA labeled with fluorescein at the 5’ end; red, 40 mer ssDNA labeled with fluorescein at the 5’ end that has been bound by Rad51 to form the presynaptic filament; green, dsDNA consisting of 40 mer ssDNA labeled with fluorescein at the 5’ end and 40 mer ssDNA labeled with rhodamine at the 3’ end; purple, complex of Rad51 and dsDNA consisting of 40 mer ssDNA labeled with fluorescein at the 5’ end and 40 mer ssDNA labeled with rhodamine.
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Ito, K., Murayama, Y., Takahashi, M. et al. Two three-strand intermediates are processed during Rad51-driven DNA strand exchange. Nat Struct Mol Biol 25, 29–36 (2018). https://doi.org/10.1038/s41594-017-0002-8
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DOI: https://doi.org/10.1038/s41594-017-0002-8
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