Structural Basis of Homology-Directed DNA Repair Mediated by RAD52

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Structural Basis of Homology-Directed DNA Repair Mediated by RAD52 Mika Saotome, Kengo Saito, Takeshi Yasuda, Hideaki Ohtomo, Shusei Sugiyama, Yoshifumi Nishimura, Hitoshi Kurumizaka, Wataru Kagawa  iScience  Volume 3, Pages 50-62 (May 2018) DOI: 10.1016/j.isci.2018.04.005 Copyright © 2018 The Author(s) Terms and Conditions

iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 “Wrapped” Configuration of the ssDNA Around the Oligomeric RAD52 Ring (A) Side view of the RAD52-ssDNA complex. To clearly depict the inner DNA binding site, the oligomeric RAD52 ring is shown in a surface representation (light blue). The ssDNA (red stick representation) is bound deeply within the cleft of RAD52 and occupies most of the space inside the groove. (B) The RAD52-ssDNA complex, viewed down the central channel of the ring. The 40-nucleotide ssDNA (5′ to 3′ counterclockwise orientation) spans across 10 RAD52 subunits. Each subunit is colored differently. (C and D) Two views of three consecutive RAD52 subunits and the 12-nucleotide ssDNA region that spans the subunits. (E) A close-up view of the electrostatic interactions of K152, R153, and R156 with the phosphate backbone of the ssDNA. Dashed lines (magenta) depict potential hydrogen bonds. (F) Hydrophobic stacking interactions that sandwich the four-nucleotide repeats. The β-hairpin structure of RAD52 (amino acid residues 52–66) is located between the four-nucleotide repeats. The ssDNA bases are sandwiched between R55 and V63 of the β-hairpin. For clarity, the DNA bases and the side chains of R55 and V63 are shown in sphere representations. See also Figures S1 and S2. iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Conformation of the ssDNA Bound to the Inner DNA Binding Site (A) The RAD52-ssDNA complex, viewed down the central channel of the ring. The four-nucleotide repeats in the 40-nucleotide ssDNA are colored differently. (B) Superimposed structures of the ssDNA repeating units depicted in (A). (C) Ideal B-form DNA. See also Figure S3. iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Putative Metal Ions Located between the Four-Nucleotide Repeats (A) Locations of the putative metal ions (pink). (B) Close-up views of the putative metal ion environment (boxed region) in (A). The putative metal ion (potassium ion) is surrounded by E140, E145, D149, and the phosphate group of the ssDNA (indicated with arrows). The table below shows the distances between the potassium ion and the potential coordinating ligands (side chain oxygen atoms from the acidic amino acid residues or oxygen atoms from the phosphate backbone of the ssDNA). The distances are averages over the 10 potassium ion-ligand interaction sites. iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 “Trapped” Configuration of ssDNA between Two RAD52 Rings (A) Crystal structure of ssDNA bound to the outer DNA binding site of RAD52. The ssDNA (red) is “trapped” between two RAD52 rings (shown in a light blue surface representation). A schematic diagram of the complex is shown in the upper right. (B and C) Close-up views of the ssDNA (boxed region) in (A). The helical structure of the ssDNA is stabilized by K102 and K133, which associate with the helical groove of the ssDNA. See also Figures S4 and S5. iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 Accumulation of the Full-Length RAD52 Protein on ssDNA Is Promoted by the Outer DNA Binding Site (A) A schematic diagram of the assay for examining the ssDNA aggregation promoted by the full-length RAD52. (B and C) ssDNA aggregation activities of the wild-type and the mutant RAD52 (K102A). A circular, plasmid-sized ssDNA (ФX174, 5,386 bases) was used as the DNA substrate. The ssDNA was fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining. Error bars indicate standard deviations. See also Figure S6. iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 ITC Isotherms for the Binding of ssDNA to RAD52 The upper panel shows the raw titration data, plotted as the heat signal (microcalories per second) versus time (minutes), obtained for 24 injections (12 μL each) of the 40-mer ssDNA oligonucleotide (polydeoxythymine) into a solution containing RAD52. The lower panel shows the ITC data from the upper panel in the form of a titration isotherm, where the integrated heat responses per injection were normalized to the moles of injected ssDNA and plotted versus the total ssDNA to 11-mer RAD52 ratio. The red patches on the schematic diagrams of the RAD52 subunits indicate the mutation sites. (A) Calorimetric titration of ssDNA (200 μM) into the RAD521−212 K133A mutant (20 μM 11-mer). The continuous curve depicts the best fit of the data to a one-site model. (B) Calorimetric titration of ssDNA (100 μM) into the RAD521−212 R55A/K152A mutant (10 μM 11-mer). The continuous curve depicts the best fit of the data to a one-site model. (C) Calorimetric titration of ssDNA (100 μM) into RAD521−212 (10 μM 11-mer). The continuous curve depicts the best fit of the data to a two-site model. See also Table S1. iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 A Model for RAD52-Mediated DNA Annealing Close-up views of the RAD52-ssDNA interactions leading to the homology search. Initially, both cDNA strands (colored blue and magenta) are “trapped” by RAD52 rings. The ssDNA moves into the inner DNA binding site of either one of the RAD52 rings that is “trapping” the ssDNA, based on the 5′ to 3′ orientation. The magenta-colored ssDNA is bound to the bottom RAD52 ring, whereas the blue-colored ssDNA is bound to the top RAD52 ring. The RAD52 rings accommodating the ssDNA associate with each other to facilitate the homology search. See also Figures S7 and S8. iScience 2018 3, 50-62DOI: (10.1016/j.isci.2018.04.005) Copyright © 2018 The Author(s) Terms and Conditions