separately with 2 mM of TmMutS2, TmMutS2DSmr, TmMutS2- Smr, and another Smr homologue (B3bp-Smr) in 20 mM Tris- HCl (pH 7.5), 150 mM KCl, 10 mM MgCl2, and 1 mM DTT at various temperatures ranging from 20uC to 80uC. In addition, various metal ions were added to the TmMutS2 DNA endonu- cleolytic reactions. The reaction mixtures were incubated at 60uC for 2 h to help establish optimized conditions. Additionally, TmMutS2 was incubated with nucleotides at various concentra- tions to verify the effect of the nucleotides (Figure 2). 2 mM of TmMutL was incubated with 2 mM TmMutS2 in the presence of 1 mM of nucleotides (ATP, ADP, and ADPnP) to study the effects of TmMutL. Each reaction was quenched by addition of DNA loading buffer (5 mM EDTA, 1% SDS, 50% glycerol, and 0.05% bromophenol blue), and then analyzed by 0.8% agarose gel electrophoresis (Figure 3). Spontaneous Mutation Frequency Analysis The spontaneous mutation frequency of the E. coli BL21(DE3) strain was estimated from the observed frequency of rifampicin- resistant mutants. Rifampicin is a bactericide, an inhibitor for the DNA-dependent RNA polymerase. Wild-type BL21(DE3) with only the pET-28aTEV vector, BL21(DE3) with the pET-28aTEV- TmrecA, and BL21(DE3) with pET-28aTEV-TmmutS2 were individually cultured in 3 mL of LB medium at 37uC for 12 h. Each culture was diluted with 150 mL of LB medium, and then shaken at 37uC for 6 h. These cultures were spread on two types of LB plates, rifampicin-free plates or rifampicin plates containing 50 mg/mL rifampicin. The plates were incubated at 37uC for 24 h. The frequencies of rifampicin mutants per 108 cells were calculated from the number of colonies formed on the rifampicin- containing and rifampicin-free LB plates (Figure 4A). In vitro DNA Homologous Recombination Assay The reaction mixtures containing 1 mM M13 phage single- stranded DNA, 1 mM SSB, 10 mM TmRecA, 50 mM Hepes- KOH (pH 7.4), 10 mM MgCl2, 2 mM ATP, 1 mM DTT, and an ATP generation system (10 mM phosphocreatine and 10 U/mL phosphocreatine kinase) were pre-incubated at 37uC for 10 min [53]. For initializing the DNA strand exchange reaction, 2 mM EcoRI-linearized M13 phage dsDNA (replication form) was added to the mixtures. Various amounts of TmMutS2 were applied, and the mixture was incubated at 37uC for 1 h. The reactions were terminated by the addition of 25 mM EDTA and 0.1% SDS after a 1 h incubation. The DNA from the reaction was analyzed with a 0.8% agarose gel stained with 0.0001% ethidium bromide (Figure 4B). SAXS Data Collection and Processing SAXS data were collected at the BL45XU beamline of the RIKEN Institute, SPring-8 (Japan). To obtain SAXS data for apo- TmMutS2 and its complexes with ADP, ADPnP, linear dsDNA, and FWJ-DNA, 29 mM of TmMutS2 was incubated with 1 mM of nucleotides and 0.15 mM DNA at 45uC for 30 min, respectively. In the case of B3bp-exSmr (residue 1618-1770 of B3bp), 100 mM of the protein was used. The sample buffer consisted of 20 mM Tris-HCl (pH 7.6), 200 mM KCl, 10 mM DTT, 1 mM EDTA, and 5% glycerol. For minimizing aggregation, all reactants were centrifuged for 10 min immediately before data collection. The parameters for X-ray scattering were as follows: incident wavelength, 0.9 A˚; photon flux, 261011 photon/sec; distance between the XR-II+CCD detector and the sample chamber, 2149.7 mm; the detector channel, S = 4psinh/l (2h: scattering angle, l: wavelength); the calibration standard, BSA; and the S Functional and Conformational Analysis of TmMutS2 range of data acquisition was from 0.006 to 0.23 A˚–1 with 100 s of exposure time. SAXS Data Analysis The initial scattering data, I(S), were scaled and subtracted from the buffer scattering value (Figure 5). Variations of the amplitude were observed in the high S range. To ascertain variations, the scattering data of the diverse conditions were linearly extrapolated. In addition, the Rg were calculated using a Guinier approximation as implemented in a PRIMUS program [54,55]. The Guinier approximation assumes that the intensity at a very small angle is represented as I(S) = I(0)exp(–4p2Rg2S2/3), where the forward scattering, I(0), is proportional to the molecular weight. About 60% points were selected to calculate the Rg values because the limit value of sRg was set as 1.3. The P(R) values were calculated from the entirety of the scattering data using an indirect Fourier transform method as implemented in the GNOM program [56]. The Dmax and Rg values, which were different from the Rg calculated by the Guinier approximation, were estimated by the P(R) function. Additionally, Rhydro was determined using a DLS analysis. The measurement was carried out in buffer containing 20 mM Tris-HCl (pH 7.6), 200 mM KCl, 10 mM DTT, 1 mM EDTA, and 5% glycerol. The calculation used a Debye-Einstein- Stokes equation: D/kBT/3gD (kB, Boltzman constant; T, absolute temperature; g, viscosity of the dispersing medium; D, apparent diffusion coefficient). Refined Modeling and Superimposition Ab initio modeling was conducted using the DAMMIN program [27] with default settings, assuming that a particle had an unknown shape bias and two-fold symmetry (Figure 6). DAMMIN represents the particle shape with a collection of densely packed beads inside a sphere with Dmax. Prior to superimposition, the ATPase and Smr domains of TmMutS2 were automatically compared with the defined structures of other proteins deposited in the PDB, and then modeled using SWISS-MODEL ver. 8.05 [57]. The low-resolution shapes of the intact and complex forms of TmMutS2 were aligned with the known structures of TaqMutS- ATPase (PDB No. 1EWQ), B3bp-Smr (PDB No. 2D9I), and the modeled structures of the TmMutS2-ATPase and TmMutS2-Smr domains using SUPCOMB [58] as shown in Figure 7 and 8. Refined rigid body models were generated using PyMOL [59]. Supporting Information Figure S1 Domain and sequence alignments for TmMutS2. (A) The schematic domain representations of TmMutS2, MutS2 homologues, MutS, and the Smr single domain. The conserved domains for all proteins were identified by sequence alignment analysis and are represented by the colored regions. All sequences were obtained from Genbank: TmMutS2, Thermotoga maritima MutS2 (NP_229083.1); HpMutS2, Helicobacter pylori MutS2 (NP_223283); AtMutS2, Arabidopsis thaliana MutS2 homologue (NP_200220); HsMSH4, Homo sapiens MutS homo- logue (MSH4, NP_002431.2); HsMSH5, Homo sapiens MutS homologue (MSH5, NP_002432.1); EcMutS, Escherichia coli MutS (NP_417213); and EcYdaL, Escherichia coli YdaL (NP_415856.1). DNA binding (blue) and ATPase (red) indicate the DNA-binding and ATP-hydrolytic domains, respectively. Mismatch (grey) and Smr (green) indicate the mismatched DNA-binding region and the Smr domain, respectively. (B) Sequence alignment of the DNA binding domain among TmMutS2 and other MutS2 homologues. (C) Sequence alignment of the ATPase domain among TmMutS2 PLoS ONE | www.plosone.org 13 April 2012 | Volume 7 | Issue 4 | e34529