(50 mM Hepes-NaOH, pH 7.5, 100 mM NaCl, 1 mM dithio- threitol (DTT), 5 mM MgCl2, 0.5 mM ethylenediaminetetraacetic acid (EDTA), and 5% glycerol). They were eluted in buffer II with 1 M NaCl as a linear gradient (flow rate: 1 to 2 mL/min). The final purification was carried out using Superdex 200 HR (TmMutS2, TmMutL, and TmRecA) and Superdex 75 HR (B3bp-exSmr and B3bp-Smr) size exclusion columns (GE Healthcare, Denmark) pre-equilibrated with buffer III (25 mM Hepes-NaOH; pH 7.5; 100 mM NaCl for B3bp-exSmr and B3bp-Smr or 200 mM NaCl for TmMutS2, TmMutL, and TmRecA; 5 mM MgCl2; 0.5 mM EDTA; and 5% glycerol). The highly purified proteins were concentrated using an Ultracel Amicon YM-10 (Millipore, USA), and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence Alignment Analysis The multiple sequence alignments for the TmMutS2-related proteins such as TmMutS2, MutS2 homologues, TmMutS2-Smr, and Smr homologues were performed using CLUSTALW [48] and ESPript version 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ ESPript.cgi) [49] with the default parameter settings (Figure S1). Measurements of ATPase Activity TmMutS2 was incubated with 750 pmol of a-32P-labeled ATP at 37 and 60uC for 30 min in a reaction solution containing 0.1 M KCl, 50 mM Tris-HCl (pH 8.0), and 5 mM MgCl2. One microliter of each reaction mixture was spotted onto a PEI-TLC plate (Figure S2). Labeled ATP and ADP were separated by developing the TLC plate in an incubation solution containing 1 M formic acid and 0.4 M LiCl [50] before detection using a bio- imaging analyzer (FLA-2000, FUJIFILM). ATP hydrolysis kinetic data were obtained for various concentrations of TmMutS2 (0.7, 1.2, and 2 mM) with 500 mM ATP. To calculate the specific ATPase activity of TmMutS2, a fluorometric real-time assay was carried out [51]. The reaction was conducted in a solution consisting of 0.1 M KCl, 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 90 mM NADH, 0.9 mM PEP, 5.3 U/mL pyruvate kinase, 7.5 U/mL lactate dehydrogenase, 0.5 mM ATP, and 400 nM TmL. The reaction was continuously monitored at 340 nm using a spectrophotometer (Shimadzu UV1601, Germany). The influ- ence of stability of TmMutS2 to ATPase activity was monitored using a DLS instrument (Zetasizer Nano Malvern, UK). It was performed with TmMutS2 in buffer III at both 37uC and 60uC for 0, 30, 60, and 90 min (Figure S2). Scanning Probe Microscopy Observation The highly purified TmMutS2 was spread on a fresh circular mica sheet (9.9 diameters; Ted Pella Inc., USA). A series of droplets was gently dispensed on the central mica surface by pipetting [52]. Sample mounting was conducted in approximately 1 min intervals as follows: 50 mL of double-distilled (dd) H2O for pre-rinsing, 50 mL of 100 mM MgCl2 for supporting affinity of the proteins, 50 mL of ddH2O for rinsing, 10 mL of TmMutS2 solution, and then three applications of 50 mL of ddH2O for post- rinsing of the mica surface. After the sample was mounted, the mica sheet was air-dried, and then inspected using SPM as shown in Figure 1A. Analytical Ultracentrifugation To verify the homogeneity of TmMutS2, the sedimentation velocity (SV) measurements were performed using an Optima XL- A analytical ultracentrifuge (BECKMAN, USA) equipped with an An60Ti rotor and a photoelectric scanner at 20uC as previously Functional and Conformational Analysis of TmMutS2 described [30]. The protein was injected into a double-sector cell with a 12-mm Epon centerpiece and quartz windows. The rotor speed was 40000 rpm, and the running time was 24 h. All parameters for the SV analysis were adjusted to standard conditions (water at 20uC). The data were analyzed by the C(s) method [31] using the ULTRASCAN program (Figure 1B). DNA Binding Assay Oligonucleotides were prepared as shown in Table S3 according to previously reported DNA sequences (1: blunt duplex, 2: 59-overhang, 3: FWJ, 4: flayed) [4], followed by heating to 95uC, and then cooling to 25uC for 6 h to anneal each strand. A polynucleotide kinase (TAKARA, Japan) was used to label the 59- end of various DNAs (10 nM) with [c–32P]ATP (30 nM). Labeled DNA was purified by phenol/ethanol precipitation. TmMutS2 (0, 0.5, 1.0, and 2.0 mM) was incubated with unlabeled and labeled DNAs (1 mM) at 37uC for 1 h, separately in binding buffer (25 mM Hepes-NaOH, pH 7.5, 200 mM NaCl, and 0.5 mM EDTA). To observe the effect of ATP, 1 mM ATP was combined with TmMutS2 (2.0 mM). All results were analyzed using an electrophoretic mobility shift assay with a BAS 2000 bio-image analyzer, and quantified using the Image Gauge program (Figure 1C). Proteolytic Digestion Limited proteolytic analysis using chymotrypsin was carried out in reaction buffer (25 mM Hepes-NaOH, pH 7.5, 200 mM NaCl, 5 mM MgCl2, and 0.5 mM EDTA) at 37uC to verify conforma- tional discrepancies among the TmMutS2 complexes. The protease-to-TmMutS2 ratio was optimized to 1:1000 (w/w) for time-resolved digestion with 5 mg of initial TmMutS2. To observe the digestion patterns of TmMutS2, 0.3 mM ADP, 1 mM ADPnP, and 1 mM FWJ-DNA were added to respective proteolytic reaction mixtures. Only FWJ-DNA was employed for proteolytic analysis due to its high binding affinity with TmMutS2. Aliquots of the each total reaction mixture were withdrawn at specific time intervals. At each time point, 16 mL of each reaction mixture was combined with 4 mL of protein-staining buffer, followed by boiling and analysis by 15% SDS-PAGE (Figure 1D). Size exclusion Chromatographic Analysis TmMutS2 (10 mM) was pre-incubated with two types of nucleotides, 1 mM ADP and 1 mM ADPnP at 37uC for 2 h. Apo- and complex-protein forms along with nucleotides were loaded into the Superdex 200 HR size exclusion column pre- equilibrated with buffer III at 20uC to validate the conformational transitions of the TmMutS2 complexes. Eluted proteins were characterized in terms of Kav value: Kav = (Ve–Vo)/(Vt–Vo), where Ve is the elution volume of each protein, Vo is the void volume of the column, and Vt is the total bed volume determined by the elution of blue dextran 2000 (Sigma). Molecular weight standard proteins (Sigma, USA), apo ferritin (440 kDa), catalase (232 kDa), and bovine serum albumin (BSA, 66 kDa in the monomer form) were used for size calibration (Figure 1E). Measurements of Endonucleolytic Nicking Activity To determine whether TmMutS2 possessed nicking endonu- clease activity, fresh pBlueScript KS (II) (Stratagene, USA) plasmid DNA was utilized. First, to isolate the supercoiled circular plasmid DNA form among the several types of DNA, an agarose gel extraction method was employed. Then, to compare nicking endonuclease activities among MutS2 homologues, supercoiled plasmid DNA (1.5 mM, approximately 3 kbp size) was incubated PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e34529