Volume 10, Issue 2, Pages 359-371 (August 2002) Crystal Structure of the Homologous-Pairing Domain from the Human Rad52 Recombinase in the Undecameric Form  Wataru Kagawa, Hitoshi Kurumizaka, Ryuichiro Ishitani, Shuya Fukai, Osamu Nureki, Takehiko Shibata, Shigeyuki Yokoyama  Molecular Cell  Volume 10, Issue 2, Pages 359-371 (August 2002) DOI: 10.1016/S1097-2765(02)00587-7

Figure 1 The Structure of Rad521-212 (A) Ribbon diagram of the undecameric ring of Rad521-212, viewed down the central channel from the top of the domed cap (Figure 2A). The eleven monomers, each colored differently, have a rotational symmetry axis that runs down the center of the ring. The diameter of the ring is about 120 Å, and that of the central hole is about 25 Å at the narrowest point and 50 Å at the widest point. (B) A Rad521-212 monomer in (A) is viewed from the rotational 11-fold axis. The domed cap region is colored in blue and magenta, and the stem region is colored in gray. (C) The Rad521-212 monomer in (B) is viewed from the left. Molecular Cell 2002 10, 359-371DOI: (10.1016/S1097-2765(02)00587-7)

Figure 2 The Side View and the Bottom View of the Mushroom-like Structure of the Undecameric Rad521-212 Ring (A and C) The domed cap region is colored in blue and magenta, and the stem region is colored in gray. (B and D) The solvent-accessible surface, colored according to the local electrostatic potential from −12 kBT−1 (red) to +12 kBT−1 (blue). Molecular Cell 2002 10, 359-371DOI: (10.1016/S1097-2765(02)00587-7)

Figure 3 The Secondary Structure of the Rad521-212 Monomer (A) The conserved sequences of the N-terminal regions of Rad52 from human, mouse (Muris et al., 1994), chicken (Bezzubova et al., 1993), and yeast (Adzuma et al., 1984; Milne and Weaver, 1993; Ostermann et al., 1993; van den Bosch et al., 2001a), and also from Rad59 (Bai and Symington, 1996; van den Bosch et al., 2001b) are aligned with the secondary structure elements of Rad521-212. Conserved amino acid residues among these proteins are colored in orange. The secondary structure elements in the stem region are colored in gray and those in the N- and C-terminal parts of the domed cap region are colored in blue and in magenta, respectively. (B) Abbreviated topology diagram showing the fold of the Rad521-212 monomer. Rods and arrows indicate α helices and β strands, respectively. Molecular Cell 2002 10, 359-371DOI: (10.1016/S1097-2765(02)00587-7)

Figure 4 The DNA Binding Groove of Rad52 (A) Stereo representation of a close-up view of the DNA binding groove. (B) Amino acid residues of Rad521-212 that were replaced with alanine for DNA binding studies. (C) Residues that affected the ssDNA binding and the dsDNA binding most severely are colored in gold and sky blue, respectively, and are mapped on the Rad521-212 ring, viewed from below. A Rad521-212 monomer is colored in magenta. (D) Stereo representation of the 2|Fo| - |Fc| electron density map of the labeled DNA binding residues shown in (C), contoured at 1.0 σ. Molecular Cell 2002 10, 359-371DOI: (10.1016/S1097-2765(02)00587-7)

Figure 5 DNA Binding, Ternary Complex Formation, and D Loop Formation by Rad521-212 All assays were analyzed by 1% agarose gel electrophoresis. (A) ssDNA binding by Rad521-212 and its point mutants was analyzed by incubating 0.5, 1, 1.5, or 2 μM of the proteins with a 50-mer ssDNA (1 μM). (B) dsDNA binding by Rad521-212 and its point mutants was analyzed by incubating 0.5, 1, or 1.5 μM of the proteins with a negatively supercoiled plasmid DNA (15 μM, 3218 bp). Graphical representations of the ssDNA and dsDNA binding by the R55A and Y65A mutants (C and E) and by the K152A, R153A, and R156A mutants (D and F). Ternary complex (G) and D loop (H) formation by Rad521-212. The indicated amounts of Rad521-212 were preincubated with a 50-mer ssDNA (1 μM) for 6 min, followed by the addition of a superhelical dsDNA (15 μM, 3218 bp) to initiate the reaction. The order of the DNA substrate addition is switched in lanes 11–15 (G) and in lanes 6–10 (H). Molecular Cell 2002 10, 359-371DOI: (10.1016/S1097-2765(02)00587-7)

Figure 6 Sedimentation Equilibrium of the Full-Length Rad52 (A) and Rad521-212 (B) Proteins For the molecular weight analysis, data were fit to an ideal, single component model. A model of the Rad52 heptameric ring, viewed from the top (C) and from an angle (D). The space between the top portions of the β sheet in the stem could accommodate two β strands (colored in red). Molecular Cell 2002 10, 359-371DOI: (10.1016/S1097-2765(02)00587-7)