Comparison of hRad52 structure and homology model of Redβ.

<p>(A) Structure of undecameric N-terminal hRad52_1–212 (PDB accession 1KN0) showing residues involved in ssDNA binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869-Kagawa2" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869-Lloyd1" target="_blank">[38]</a>. The structure is colored as follows: residues 25–78 (part of the “domed cap”) in light blue, residues 79–156 (“stem structure”) in green, residues 157–220 (part of the “domed cap”) in dark blue. The N- and C- termini are also indicated. (B and C) Homology-based model showing sequence conservation between hRad52 and Redβ. (B) The hRad52_1–212 oligomeric ring structure is shown, with the stem region in green, and the flanking regions in pink. One subunit is rendered in surface view and colored according to sequence conservation between Redβ and Rad52 family proteins based on the alignment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone-0078869-g001" target="_blank">Figure 1</a> (yellow to red, least to highest degree of conservation), with the aid of the ConSurf server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869-Ashkenazy1" target="_blank">[49]</a>. The basic groove predicted to bind ssDNA is indicated by a cyan dotted line. (C) Two views of a subunit of the 1KN0 structure (truncated to residues 25–177 for clarity) and colored according to the degree of conservation with respect to Redβ as in (B). The two conserved patches and predicted locations of the Redβ residues probed in this study are indicated. Residue V77 is buried and is therefore not displayed.</p

Binding of Redβ WT and mutant proteins to sequentially added complementary DNA strands.

<p>Gel retardation assays were performed using varying concentrations of Redβ (0.1, 0.3, 0.9, 2.7 µM) and 5 nM each of sequentially added complementary DNA strands. Images of the actual gels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869.s002" target="_blank">Figure S2</a>.</p

Characterization of the oligomeric structure of Redβ mutant proteins.

<p>(A) Native PAGE analysis of Redβ protein variants. Proteins (10 µg per lane) were separated on a 5–20% gradient polyacrylamide gel and visualized by staining with Coomassie Blue. (B) Size-exclusion chromatography of Redβ mutants. Proteins (10 µl of 6 mg/ml) were applied to a Superdex 200 5/150 GL gel filtration column in 20 mM potassium phosphate pH 6.0, 10 mM MgCl₂, 0.15 M NaCl. Molecular masses were estimated by constructing a calibration curve from known molecular mass standards: ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa) and ferritin (440 kDa).</p

Visualization of Redβ WT and mutant protein oligomers by TEM.

<p>Images were obtained for proteins (2 µM) alone (A) or in the presence of 1 µM 50 nt ssDNA (B). The scale bars represent 50 nm.</p

Molecular weights of GyrA-CTD proteins calculated from their amino acid sequences and elution volumes from analytical size exclusion chromatography.

<p>Molecular weights of GyrA-CTD proteins calculated from their amino acid sequences and elution volumes from analytical size exclusion chromatography.</p

Comparison of Redβ, Rad52 and Sak N-terminal domains.

<p>Conserved residues are highlighted and colored according to the Clustal X scheme, with color intensity denoting the degree of homology between the three SSAP groups. The green boxed region indicates the stem structure of Rad52 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869-Kagawa2" target="_blank">[32]</a>; secondary structure elements (β-β-β-α) in this region are highlighted. The blue boxed regions correspond to the conserved part of the domed cap region in the hRad52 N-terminal structure, while pink boxed regions indicate alternative conserved stretches in Redβ that flank the putative stem structure. Residues of Redβ mutated to alanine in this study are marked with blue circles and numbered accordingly. Residues of hRad52 that have been experimentally shown to be involved in ssDNA binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869-Kagawa2" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869-Lloyd1" target="_blank">[38]</a> are indicated by red circles. Sak SSAP from phage ul36 and related sequences are also shown. Abbreviations and UniProt accession numbers: <i>Ep_beta</i>, Redβ from Enterobacteria phage λ (<u>P03698</u>); <i>Mn_beta</i>, bacteriophage recombinase from <i>Mannheimia haemolytica</i> (<u>A7JWQ9</u>); <i>Yp_beta</i>, DNA recombination protein from <i>Yersinia pestis</i> biovar Orientalis str. (<u>A4IUY1</u>); <i>Vc_beta</i>, putative DNA recombination protein from <i>Vibrio cholerae</i> (<u>Q8KQW0</u>); <i>Ip_beta</i>, phage recombination protein from Iodobacteriophage φPLPE (<u>B5AX97</u>); <i>Bp_beta</i>, putative phage recombination protein from <i>Burkholderia pseudomallei</i> strain 668 (A3NM00); <i>Ab_beta</i>, phage recombination protein Bet from <i>Acinetobacter baumannii</i>; <i>Hs_Rad52</i>, Rad52 homolog from <i>Homo sapiens</i> (<u>P43351</u>); <i>Sc_Rad52</i>, Rad52 from <i>Saccharomyces cerevisiae</i> (<u>P06778</u>); <i>Sp_Rad22</i>, Rad22 protein from <i>Schizosaccharomyces pombe</i> (<u>P36592</u>); <i>Sp_Rti1</i>, Rti1 protein from <i>S. pombe</i> (<u>O42905</u>); <i>ul36_Sak</i>, putative translation initiation factor from <i>Lactococcus</i> phage ul36 (<u>Q9MC33_9CAUD</u>). The three additional sequences in the Sak family are identified by GI accession numbers. The alignment display was generated using Jalview <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078869#pone.0078869-Waterhouse1" target="_blank">[48]</a>.</p

Binding of Redβ WT and mutant proteins to ssDNA.

<p>(A) Gel retardation assays showing binding of Redβ WT and mutant proteins to ssDNA. Varying concentrations of Redβ (0, 0.09, 0.19, 0.38, 0.75, 1.5, 3, 6, 12.5, 25, 50, 100 µM) were mixed with 10 nM 5′ Cy3-labeled 50 mer oligonucleotide as described in the Materials and Methods. Samples were fixed with 0.1% glutaraldehyde prior to separation on 6% PAGE. (B and C) DNA binding analysis based on quantification of the gels in (A). The percentage ssDNA bound by Redβ proteins at 3 µM is presented as a bar graph in (C).</p

Gyrase β-pinwheel blade motif alignments.

<p>Aligned sequences of the region corresponding to the first (<b>A</b>), the second (<b>B</b>) and the sixth (<b>C</b>) β-pinwheel blade motifs of prokaryotic gyrases. Red, yellow and green elements above the sequences represent α-helices, β-strands and coils in the structure of <i>X</i>. <i>campestris</i> GyrA CTD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref038" target="_blank">38</a>], respectively (PDB code: 3L6V chain A, as viewed by PyMOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref039" target="_blank">39</a>]). Grey-filled triangle indicates the trypsinolysis site for EcGyrA (Arg571) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref040" target="_blank">40</a>] whereas unfilled triangles indicate those for PfGyrA (Arg854, Lys856 and Lys862). The putative coiled-coil of PfGyrA predicted by the SMART server is shown in red below the sequences in A. Annotations and smaller filled arrows underneath the sequences in A and B indicate the elements found in PfGyrA that are conserved with prokaryotic GyrA. <b>D.</b> X-ray crystal structure of <i>X</i>. <i>campestris</i> GyrA (PDB code: 3L6V [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref038" target="_blank">38</a>]) shown in cartoon format with secondary structure elements coloured as described for A-C. Each of the six blades are numbered. The hydrophobic region of blade 1 is coloured orange and shown in stick format. The conserved S/T residue of blade 1 is shown in blue, in stick format and the conserved G of blade 1 is shown in cyan, in stick format.</p

Proportion of secondary structures calculated from CD-spectra using the Dichroweb server [32].

<p>Proportion of secondary structures calculated from CD-spectra using the Dichroweb server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref032" target="_blank">32</a>].</p

Limited trypsinolysis of PfGyrA-CTD.

<p>Protein digests were visualised as a function of time. Time elapsed in minutes after initiation of reaction is indicated along the top of the gel. Decreases in band intensities of the original proteins (black triangles) are associated with reciprocal increases in band intensities of the fragments (white triangles). Edman degradation has identified the following amino acid sequences from the denoted fragments. Ec1, 572-IKEED-576; Ec2 1-<u>GSH</u>ME-2 [underlined Gly-Ser-His residues derived from pET28a(+)]; Pf1, 855-LKFND-859 (also 857-FNDLQ-861 and 863-GNEQE-867 at lower intensities); Pf2, MKDHL (derived from the N-terminal His-tag).</p