Characterization of Biochemical Properties for TmMutS2

Prior to the verification of characteristics for TmMutS2, its sequence was analyzed via comparisons with homologues through BLAST searches (Figure S1). Sequence alignment analysis indicated common features between TmMutS2 and other MutS2 homologues via the conserved DNA-binding and ATP-hydrolytic domains (Figure S1). TmMutS2, as well as other MutS2 homologues such as H. pylori MutS2, E. coli YdaL, and Thermus thermophilus MutS2 [13], [17]–[19], contain a conserved C-terminal domain of approximately 100 to 250 amino acid residues, which is identified as the Smr domain. They also lack a conserved N-terminal region as a recognition site for mismatched DNA, whereas E. coli MutS does not possess the Smr domain but does have the N-terminal recognition site. Therefore, it is expected that TmMutS2 has the Smr-related functions but lacks the identification activity for mismatched DNA.

A highly conserved Walker’s A-type nucleotide-binding motif in TmMutS2 (Figure S1) represents the ATPase domain as previously reported for T. thermophilus MutS2 [17]. To examine the ATPase activity of TmMutS2, an ATPase assay was carried out in two ways using highly purified TmMutS2 (89 kDa, Figure S2). Polyethyleneimine-cellulose thin-layer chromatography (PEI-TLC) analysis revealed that TmMutS2 had a higher ATPase activity at 60°C compared to 37°C, and a fluorometric analysis also indicated a two-fold increase of activity at 60°C compared with 37°C (Figure S2). To clarify the effect of instability of TmMutS2, dynamic light scattering (DLS) measurements at both 37°C and 60°C with time dependence (0, 30, 60, and 90 min) were conducted. As shown in Figure S2, the average size of TmMutS2 was invariant despite variations of temperature and time. This indicates that the stability of the protein was maintained during the measurement of ATPase activity. Therefore, the ATPase activity discrepancy is only influenced by temperature. In addition, double-stranded DNA (dsDNA) slightly affects the ATP-hydrolytic activity of TmMutS2 as represented in Figure S2. These results indicate that TmMutS2 possesses thermo-active ATPase activity that functions in a DNA-dependent manner [17].

The multimeric status of TmMutS2 was confirmed by two ways. In the scanning probe microscopy (SPM) observation, the shape of the TmMutS2 molecules is a two-fold symmetric form, implying that TmMutS2 may form a dimer (white dash line) as shown in Figure 1A. This dimeric status was further determined by analytical ultracentrifugation. The distribution of the sedimentation coefficient was obtained using the C(s) method [30], [31]. A single narrow peak with a mean average sedimentation coefficient of 5.2 (±0.2)×10^–13 s indicated that TmMutS2 exists as a homogeneous dimer (Figure 1B).

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Figure 1. Biochemical properties of TmMutS2.

(A) The SPM image of the TmMutS2 protein. Height is indicated by the colors dark (0 nm) and light (9.7 nm) brown. The shape of TmMutS2 molecules on the mica looks appears to be a two-fold symmetric form (the white dashed line). (B) Sedimentation velocity analysis of TmMutS2. The absorbance at 280 nm was recorded every 4 min (upper plot). The lower plot is the C(s) analysis of TmMutS2. (C) DNA binding activities of TmMutS2 in the absence or presence of ATP (1 mM). TmMutS2 concentration is varied from 0 to 2 µM. DNA substrates 1, 2, 3, and 4 are the linear, overhanging, four-way junction, and flattened linear flayed forms, respectively. The right plot indicates the % bound complex, represented by the dashed line boxes (1′–4′), in the gel. (D) Limited digestion by α-chymotrypsin. Lane 1 represents the protein molecular weight marker. Lane 2 is the protease-untreated TmMutS2 (5 µg). TmMutS2 was digested in the presence of 0.3 mM ADP (lanes 5–6), 1 mM ADPnP (lanes 7–8), and 1 µM FWJ-DNA (lanes 9–10). The arrow indicates the N-terminal truncated form (residue 117 to 757) and the asterisk marks the N-terminal fragment. (E) Size exclusion chromatographic analysis. Blue and red peaks are the detection profiles of absorbance at 280 and 254 nm, respectively. The molecular sizes of TmMutS2 and its complexes with nucleotides were estimated from their elution volumes (V_e). The protein standards were used for the standardization of molecular weights using K_av = (V_e–V_o)/(V_c–V_o), where V_c is the geometric column volume and V_o is the void volume that is calculated by Blue dextran (2000 kDa).

https://doi.org/10.1371/journal.pone.0034529.g001

It is well known that MutS2 homologues interact with various forms of DNA [15]–[17]. In order to ascertain the DNA binding activity of TmMutS2, gel electrophoresis was carried out. TmMutS2 more strongly bound to four-way junction (FWJ) and flayed DNA (a fork structure) compared to linear and overhang DNA (Figure 1C). The addition of 1 mM ATP decreased the gel-shifting yield of TmMutS2-DNA complexes (particularly FWJ-DNA), thus suggesting that ATP acted as an obstacle with DNA for binding with TmMutS2. In addition, super-shifted bands on the gel were not detected after adding ATP. Hence, it is obvious that nucleotides play a crucial role in the binding of DNA to TmMutS2 even absent any alteration of multimeric status of the complexes.

The binding of nucleotides may affect the conformation of the protein. Proteolytic digestion analysis by α-chymotrypsin indicated conformational transitions of the TmMutS2-nucleotide and -DNA complexes (Figure 1D). Only FWJ-DNA was selected for proteolytic digestion due to it’s the strongest binding affinity with TmMutS2 among several types of DNA. TmMutS2 with nucleotides (particularly nonhydrolytic ATP; ADPnP) revealed faint digested bands rather than apo or complex forms with FWJ-DNA, providing clear evidence that TmMutS2 underwent conformational changes in a nucleotides-activated manner. Size exclusion chromatographic (SEC) analysis was also employed to verify the effects of nucleotides on conformation. Because of insignificant differences of digested bands between apo and FWJ-DNA complex forms, TmMutS2-nucleotide complexes were only applied to SEC analysis. As represented in Figure 1E, the elution profile of the native TmMutS2 exhibited only one broad peak ranging from 160 to 190 kDa, implying a dimeric TmMutS2 without any aggregate form. The results support the SPM and sedimentation velocity analysis. However, the increased elution volumes, 11.13 mL for TmMutS2-ADP and 11.42 mL for TmMutS2-ADPnP complexes, were noted (compare with 11.05 mL for apo-TmMutS2). These results suggest that there is shrinkage of TmMutS2 after nucleotide binding. Therefore, we suggest that the structure of TmMutS2 assumed a compacted form in the presence of nucleotides, particularly non-hydrolyzable ADPnP.

Identification of Nicking Endonuclease Activity for TmMutS2

It has been widely known that the nicking endonuclease activity of MutS2 homologues alters closed circular dsDNA leading to an open circular form under variable physical conditions [17], [18], [32]. Thus, it was expected that this TmMutS2 would also have inherent endonuclease activity. To demonstrate the DNA nicking endonuclease activity of TmMutS2, two types of protein, apo-TmMutS2 and TmMutS2-Smr, were incubated with freshly prepared plasmid DNA. These reactants were then analyzed by 0.8% agarose gel electrophoresis. First, various cations were applied to TmMutS2 for optimizing its nicking endonuclease activity (Figure 2A). The Mg²⁺ ion was determined to be the most effective cation to promote incision of plasmid DNA by TmMutS2, so it was applied to all reactions. In the absence of Mg²⁺, TmMutS2 presented lower nicking endonuclease activity (Figure 2B, right two lanes). This reaction was carried out in the buffer with 0.1 mM EDTA to eliminate trace of Mg²⁺. The nicking endonuclease activity of TmMutS2 increased in proportion to the tested temperature, ranging from 20°C to 60°C (left three lanes). In addition, TmMutS2-Smr also exhibited temperature-dependent endonuclease activity, but activity decreased in harsh conditions such as temperatures of 80°C (Figure 2B, from 6 th lane to 9 th lane). TmMutS2-Smr has higher activity compared to native TmMutS2 (Figure 2B, lower plot), indicating that endonuclease activity was confined to TmMutS2-Smr, which corresponded to the characteristics of other MutS2 homologues. A further experiment substantiated this fact as described in Figure S3. TmMutS2 and TmMutS2ΔSmr were incubated with supercoiled circular dsDNA (scDNA). TmMutS2ΔSmr presented no nicking endonuclease activity, suggesting that the Smr domain of TmMutS2 played a major role in the incision of the scDNA. TmMutS2-Smr, as well as human B3bp-Smr possessed a high level of nicking endonuclease activity, as expected (Figure S3). Therefore, it was anticipated that several species without endonucleases, such as MutH, possess alternative proteins that have the active Smr domain to provide the endonuclease function.

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Figure 2. Nicking endonuclease activity of TmMutS2.

(A) The effects of various cations on the nicking endonuclease activity of TmMutS2. The cations were used at a concentration of 10 mM. The nick, l, and sc indicate the nicked, linearized, and supercoiled open circular DNA forms, respectively. (B) A mixture containing 2 µM TmMutS2/TmMutS2-Smr and 1.5 µM plasmid DNA (linear size, 3 kbp) was incubated at temperatures ranging from 20°C to 80°C in the presence or absence of Mg²⁺ (upper plot). In the middle of the gel, M indicates the DNA size marker. The lower plot presents the percentage yields of the nicked form. (C) Different activities of DNA digestion in the presence of ATP (lanes 3 to 7), ADP (lanes 8 to 12), and ADPnP (lanes 13 to 17). Lane 1 and 2 are TmMutS2 untreated- and treated-plasmid DNA bands in the absence of theh nucleotide at 60°C. Each nucleotide was utilized at a final concentration of 3 mM.

https://doi.org/10.1371/journal.pone.0034529.g002

Nicking endonuclease activity was influenced by nucleotides (ATP, ADP, and ADPnP). As shown in Figure 2C, ATP and ADP did not affect the nicking endonuclease activity of TmMutS2, but quite high concentrations (above 2.5 mM) of ADPnP negatively impacted the plasmid DNA digestion, indicating the binding of ADPnP caused a decrease in the TmMutS2 activity. It was predicted that the nicking endonuclease activity of MutS2 proteins may be controlled by a particular nucleotide.

Analysis of the MutL Effect on Endonuclease Activity of TmMutS2

E. coli MutL is already known for participating in the MMR system [1], and MutL homologues from other bacterial species have been recognized as the activators for endonuclease activity of their respective MutS2 proteins [17]. The addition of TmMutL to TmMutS2 also resulted in an increase in nicking endonuclease activity (Figure 3A). This was in accordance with other evidence indicating that the TmMutS2-TmMutL complex has higher dsDNA binding affinity rather than TmMutS2 alone (Figure S4). Additionally, we examined the impact of nucleotides on the endonuclease activity of the TmMutS2-TmMutL mixture. The addition of nucleotides, particularly ADPnP, caused a decrease in the nicking endonuclease activity of TmMutS2 via the binding of the nucleotides to TmMutL (Figure 3B). As shown in Figure S5, only TmMutL did not show endonuclease activity as well as dependence on the nucleotide. These results suggest that the alterable nicking endonuclease activity of TmMutS2 is negatively controlled by the interaction of TmMutL with nucleotides.

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Figure 3. The TmMutL effects on the endonuclease activity of TmMutS2.

(A) The 0.8% agarose gel indicates that TmMutL enhances nicking endonuclease activity and the DNA binding affinity of TmMutS2. Lane 1 represents TmMutS2 untreated DNA, and the B at lane 2 indicates EcoRI-treated linearized dsDNA. (B) The addition of 1 mM of nucleotide and 2 µM TmMutL to TmMutS2.

https://doi.org/10.1371/journal.pone.0034529.g003

Confirmation of the Influences of TmMutS2 on DNA Homologous Recombination

To understand the role of TmMutS2 on DNA homologous recombination, a spontaneous mutation frequency assay was carried out using TmRecA, a core protein involved in DNA strand exchange. As shown in Figure 4A, the mutation frequency of the BL21(DE3) strain with only pET-28a Tobacco Etch virus (TEV) and pET-28aTEV-TmrecA did not yield any significant difference in mutational frequency compared with the wild-type strain (1.39 (±0.03)×10^–8 and 0.91 (±0.07)×10^–8 rifampicin-resistance colonies, respectively), while incorporation of the TmmutS2 gene into pET-28aTEV resulted in an approximately 6-fold increase of the spontaneous mutation frequency (6.63 (±0.13)×10^–8). Additionally, the mutation on the Smr domain caused conspicuous decrease of the mutation frequency in comparison with wild type TmMutS2 (2.67 (±0.20)×10^–8 and 7.33 (±0.47)×10^–8 rifampicin-resistance colonies, respectively), indicating that the Smr domain plays an important role in the RecA-related recombination (Figure S6). This recombination experiment reveals that TmmutS2 may affect the RecA-related recombination and act as a dominant mutation phenotype similar to other MutS2 homologues [15].

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Figure 4. Suppression of the TmRecA-mediated DNA recombination reaction by TmMutS2.

(A) A spontaneous mutation frequency assay using the incorporation of TmmutS2 gene into the E. coli BL21(DE3) strain. Rif^R indicates rifampicin-resistance. The frequency of Rif^R (y-axis)/10⁸ cells is quantitatively analyzed by counting the number of spontaneously mutated colonies. (B) The effect of the concentration of TmMutS2 on the TmRecA-mediated DNA recombination reactions. Lanes 2, 3, 4, 5, 6, and 7 correspond to 0, 100, 200, 400, 800, and 1600 nM of TmMutS2, respectively. Nick, lds, and ssc indicate the nicked, linearized, and supercoiled forms of open circular DNA, respectively. The right plot is the quantification of the nicked forms. This plot represents the changes of TmMutS2 concentrations versus the percentage yield of nicked-form DNA. (C) To observe the influence of TmMutL in the DNA strand exchange reaction, TmRecA, TmMutS2, and TmMutL were incubated at 37°C.

https://doi.org/10.1371/journal.pone.0034529.g004

In order to inspect the effect of TmMutS2 on DNA homologous recombination in detail, TmMutS2 was incubated with TmRecA. RecA catalyzes extensive homoduplex formation between linearized DNA and single-stranded circular DNA (sscDNA) in the presence of single-stranded DNA-binding protein (SSB) and an ATP-regenerating system [33]. To further clarify the correlation between TmRecA and TmMutS2, TmMutS2 was applied to this system. Purified, closed dsDNA was completely linearized with EcoRI restriction enzyme for the apparent reaction between homologous M13 DNAs (linearized form and sscDNA). Then, linearized dsDNA (ldsDNA) and sscDNA were mixed into the reaction as described above. Faint band for the nicked form appeared in the control case due to the strand exchange reaction (Figure 4B, lane 1). The addition of TmRecA caused successful recombination between homoduplex DNA, indicating an increase in the nicked forms; new recombinant DNA consisted of sscDNA and one strand of ldsDNA (Figure 4B, lane 2). In the case of the TmMutS2 supplementation, the higher the concentration of TmMutS2, the vaguer the band of nicked dsDNA became (Figure 4B, lanes 3, 4, 5, 6, 7), indicating that TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction. As shown in Figure 4B (right plot), the yields of nicked DNA linearly decreased with increases in TmMutS2 concentration. The addition of TmMutL resulted in the loss of nicked DNA compared to TmMutS2 alone (Figure 4C). Therefore, TmMutS2 effectively inhibited RecA-mediated DNA recombination, and TmMutL also interrupted the DNA strand exchange reaction. These results strongly suggest that TmMutL may play an important role in the regulation of TmMutS2-mediated recombination activity.

Predictions of Structures using SAXS for TmMutS2 Complexes with Various Types of Nucleotides or DNA

For conformational analysis of proteins, SAXS can be directly applied to monitor the solution conformations of proteins [34], [35]. In this study, we collected SAXS data on the monodispersed solutions of TmMuS2 and its complexes with nucleotides (ADP and ADPnP) or DNA (linear dsDNA and FWJ-DNA). As shown in Figure 5A, the overlying scattering curves for the various concentrations of TmMutS2 indicate an increase in signal according to the concentration of TmMutS2, including some fluctuations at high S (Å). These signal fluctuations at the high-angle region result from structural features such as the total structure and the organization of domains [36]. Additionally, confirmation of the aggregated state of the protein can be provided by the scattering data, inspecting the linearity at the low S range using a Guinier approximation (lnI(S) vs. S²). The upper inset in Figure 5A presents the apo-TmMutS2, indicating that TmMutS exhibits monodispersity and no aggregation. For complexes of TmMutS2 with nucleotides and DNA, different signals were noted according to the partner molecules in the complex (Figure 5B). The Guinier approximation demonstrated that TmMutS2 complexes also displayed monodispersity and did not aggregate (Figure S7).

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Figure 5. SAXS analysis for the structures of TmMutS2 and its complexes with nucleotides and DNAs.

(A) SAXS scattering curves of apo-TmMutS2 at a concentration range of 1 to 5 mg/mL. The insert exhibits the Guinier approximation for the scattering data of TmMutS2 (5 mg/mL). (B) SAXS scattering curves of apo-TmMutS2 and its complexes with nucleotides (ADP and ADPnP) and DNAs (dsDNA and FWJ-DNA) as calculated by the PRIMUS program. (C) P(R) function plots estimated by GNOM program. (D) SAXS data parameters from a GNOM analysis and DAMMIN modeling. TmS2 is T. maritima TmMutS2. R_pre^* was calculated from the Guinier approximation using AutoR_g program. R_cal^§ and D_max^¶ (the maximum diameter) were calculated from the P(r) function by GNOM program. FDNA represents four-way junction DNA. D_max is the longest distance of the SAXS models as determined by the DAMMIN program. Using DLS analysis, the hydrodynamic radii (R_hydro^$) were calculated at various temperatures.

https://doi.org/10.1371/journal.pone.0034529.g005

To obtain information about the structures of proteins from SAXS data, the P(R) spectra, histograms for the existence probabilities of atoms at all distances within scattering particles, were constructed (Figure 5C). From these spectra, the radius of gyration (R_g) and the maximum particle dimension (D_max) were calculated using the GNOM program and summarized in Figure 5D (left table). R_pre and R_cal represent the values estimated from the Guinier approximation and the P(R) spectrum, respectively, and R_cal was selected as the R_g value for further discussion. In the case of TmMutS2-dsDNA and TmMutS2-FWJ-DNA, the R_g and D_max were similar to those of apo-TmMutS2. However, the distance distributions of TmMutS2-ADP and TmMutS2-ADPnP were narrower than that of apo-TmMutS2 and had a smaller D_max than apo-TmMutS2. These results indicate that the addition of nucleotides to TmMutS2 induces a smaller protein compared to the apo-TmMutS2 form. Meanwhile, the calculated R_g of apo TmMutS2 was 74.88±0.31 Å, and D_max was 250 Å. The hydrodynamic radius (R_h) was also calculated using DLS analysis in a temperature range of 25°C to 55°C. The calculation produced an averaged R_h value of 82.98 (±2.05) Å, indicating that TmMutS2 retains thermostability (Figure 5D, right table). In particular, the R_h at 45°C was 93.26 (±0.45) Å, exhibiting that the ratio of R_h to R_g is approximately 1.21, which is a similar result to the generally observed ratio (∼1.29) for known spherical molecules [37]. Thus, TmMutS2 may have a spherical configuration.

Based on the scattering data, the structures for the TmMutS2 complexes were re-constructed using the ab initio modeling program DAMMIN (Figure 6). The SAXS models of apo-TmMutS2 and its complexes presented the two-fold symmetry, consistent with the results of SPM, SEC analysis, and the sedimentation velocity measurement that verified the homogeneous dimer conformation of TmMutS2 (Figure 1A, 1B, and 1E). In addition, apo-TmMutS2 and its complexed forms seem to have two or four linking domains based on the results of the SPM analysis of the surface-distributed TmMutS2 (size range 30 to 50 nm, Figure 1A). Additionally, the TmMutS2-ADP complex retained the slightly reduced size and small valley in the central region. Unlike apo-TmMutS2 and the TmMutS2-ADP complex, the large conformational changes in TmMutS2-ADPnP were observed in the central region. The compacted TmMutS2-ADPnP complex had the smallest R_g at 62.17 (±0.21 Å) and a D_max of 180 Å, as described in above. The compactness of TmMutS2-ADPnP was already proven by the SEC analysis (Figure 1E). The TmMutS2-dsDNA and TmMutS2-FWJ-DNA complexes exhibited lengthened structures compared to the apo-TmMutS2 form. In particular, TmMutS2-FWJ-DNA formed an elongated structure resulting from large conformational changes, indicating that intermolecular interactions between the protein and FWJ-DNA may create a nearly globular structure. These results suggest that TmMutS2 assumes a flexible conformation in the presence of nucleotides and DNA.

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Figure 6. The best fitted ab initio SAXS models.

They are depicted with space-filled dummy atoms.

https://doi.org/10.1371/journal.pone.0034529.g006

Superimposition of the ATPase and Smr Models of TmMutS2 to the SAXS Models

Although much effort has been expended to define the crystal structure of TmMutS2, no one has done so. Therefore, to confirm the validity of our SAXS models, other structural information about TmMutS2 was required. As shown in Figure S8, the ATPase domain of TmMutS2 was aligned with TaqMutS. The alignment indicates that the secondary structures of the two proteins have similar features. Hence, an ATPase domain model of TmMutS2 computationally predicted from a crystal structure of TaqMutS (Protein Data Bank, PDB No. 1EWQ) was utilized as a template for structural overlapping instead of a whole crystal structure of TmMutS2 (Figure 7A). The solution SAXS models of the apo- and ADP-complexed forms could not be superimposed onto the dimeric ATPase domain of TaqMutS and the monomeric TmMutS2 because they may not form compacted structures. However, the compacted TmMutS2-ADPnP complex model was suitable for superimposition of the ATPase domains to the SAXS model (Figure 7B). In addition, whereas there is variability in the superimposition method using symmetrical differences [38], the normalized spatial discrepancy (NSD) algorithm enables fast superimposition of structural models and provides for quantitative analysis of the similarity between the models. Like error-weighted averaging discrepancy [39], NSD is characterized by fineness parameters that play the role of standard deviations for one-dimensional data sets. As shown in Table S1, the final fineness values for the superimposed SAXS structures were lower than their initial values. These tendencies make it more reliable the SAXS models.

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Figure 7. Superimpositions between the TmMutS2-ADPnP SAXS model and the ATPase structure/model.

(A) The left structure is the ATPase domain of T. aquaticus MutS (TaqS-ATPase), and the right model is the predicted monomeric ATPase domain of TmMutS2 (TmS2-ATPase). (B) Superimpositions of the TmMutS2-ADPnP model to the dimeric TaqS-ATPase structure and the TmS2-ATPase model. The predicted conformers indicate the feasible structures of the dimeric TmS2-ATPase model.

https://doi.org/10.1371/journal.pone.0034529.g007

In addition to ATPase domain analysis, the Smr domain of TmMutS2 was also characterized using the sequential homology of the Smr domain (683–757) of TmMutS2 with the Smr domain (1691–1770) of human B3bp (Figure S8). Like the preceding modeling, the TmMutS2-Smr domain was theoretically modeled from the high-resolution structure of B3bp-Smr (PDB No. 2D9I), as shown in Figure 8A. Using the TmMutS2-Smr model and the B3bp-Smr structure, the superimposition between TmMutS2-DNA (along with the dsDNA and FWJ-DNA complexes) and SAXS models was carried out (Figure 8B). Although the sizes of the B3bp-Smr structure and SAXS models seem to be different, they are similar in size. The final overlay indicated that the TmMutS2 SAXS models fit well with the respective B3bp-Smr structure and the TmMutS2-Smr model. The final fineness values for the SAXS templates and structures were lowered compared to their initial values (Table S1). In the superimposed models, both B3bp-Smr and TmMutS2-Smr are localized to the central region of the TmMutS2-DNA SAXS models, indicating that the central region of TmMutS2 is perhaps conserved but that the N-terminal region is very flexible. Interestingly, the ATPase domain (residue 310-502) of TmMutS2 is also localized to the middle location as previously described (Figure 7B). To explore the central region in more detail, these regions of the TmMutS2-DNA SAXS models were superimposed onto the B3bp-exSmr SAXS model. The good fit with the B3bp-Smr structure (Figure 8C, green mesh region) indicates that TmMutS2 has conserved domains, such as the C-terminal and DNA-binding domains. These results reveal that the addition of dsDNA and FWJ-DNA to TmMutS2 causes conformational changes in TmMutS2 (Figure 8C, circle dash-lines). These results were in accordance with the DNA-binding activities experiments (Figure 1C).

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Figure 8. Superimpositions among the TmMutS2-dsDNA/-FWJ-DNA SAXS models and the Smr structure/model.

(A) The left structure is the monomeric Smr domain of H. sapiens B3bp (B3bp-Smr), and the right model is the predicted monomeric Smr domain of TmMutS2 (TmS2-Smr). (B) Superimpositions of the TmMutS2-dsDNA/-FWJ-DNA SAXS model to the B3bp-Smr structure and the TmS2-Smr model. (C) Superimpositions of the TmMutS2-dsDNA/-FWJ-DNA SAXS model to the B3bp-exSmr SAXS model (green mesh). The B3bp-Smr structure (red) fits well with the B3bp-exSmr SAXS model.

https://doi.org/10.1371/journal.pone.0034529.g008

Cloning, Over-expression, and Purification of TmMutS2

The TmmutS2, TmmutL, and TmrecA genes were isolated from T. maritima MSB8 genomic DNA. In addition, the b3bp-Smr and b3bp-exSmr genes were isolated from Homo sapiens HeLa cell complementary DNA. All genes were amplified by PCR using specific primers as shown in Table S2. The amplified genes were inserted into the bacterial expression vector pET-28aTEV (Novagen, Germany) containing the TEV protease recognition site. In addition, the TmmutS2ΔSmr and TmmutS2-Smr (only Smr domain of TmMutS2) genes were isolated from pET-28TEV-TmmutS2, and then ligated into pET-28aTEV. The clone sequences were confirmed via automated DNA sequencing. The target proteins were over-expressed in BL21 (DE3) cells (Invitrogen, USA) grown in Luria-Bertani (LB) medium containing 50 µg/mL kanamycin at 37°C. When the UV absorbance of the cells at 600 nm reached 0.6, the protein expression was induced by the addition of 0.5 mM isopropyl-thiogalactopyranoside. The induced cells were then kept at 37°C for an additional 6 h. After the cells were harvested by centrifugation, they were resuspended in a lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5 mM β-mercaptoethanol, 5% glycerol, 1 mM phenylmethyl-sulfonyl fluoride, 1 µg/mL leupeptin, 1 µg/mL aprotinin, and 1 µg/mL benzamidine), and then disrupted by sonication. The lysate was centrifuged at 13000 rpm for 30 min at 4°C, and the supernatant was used for the further experiments. Prior to chromatography, a heat-activation step performed at 65°C for 1 h was employed for only TmMutS2, TmMutL, and TmRecA to remove the endogenous proteins extracted from the cells. In the case of B3bp-Smr, supernatants were directly applied to a nickel-nitrilotriacetic acid (Ni-NTA, GE Healthcare, Denmark) affinity column pre-equilibrated with buffer I (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5 mM β-mercaptoethanol, 5% glycerol, and 5 mM imidazole), and then eluted in buffer I with 300 mM imidazole. His-tag fragments of the purified samples were eliminated by adding TEV protease at room temperature (RT) for 8 h. To remove the untreated His-tagged proteins and the cleaved His-tag fragments, the samples were reloaded onto the Ni-NTA affinity column, and the target proteins were obtained via flow-through against buffer I with 30 mM imidazole. For further purification, each protein was individually applied to a Heparin HitrapTM HP (TmMutS2, TmMutL, and TmRecA) and a Mono S (B3bp-Smr) ion exchange column (GE Healthcare, Denmark) equilibrated with buffer II (50 mM Hepes-NaOH, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (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 α-³²P-labeled ATP at 37 and 60°C 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 µM) with 500 µM 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 µM 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 influence 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 37°C and 60°C 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 µL of double-distilled (dd) H₂O for pre-rinsing, 50 µL of 100 mM MgCl₂ for supporting affinity of the proteins, 50 µL of ddH₂O for rinsing, 10 µL of TmMutS2 solution, and then three applications of 50 µL 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 20°C as previously 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 20°C). 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: 5′-overhang, 3: FWJ, 4: flayed) [4], followed by heating to 95°C, and then cooling to 25°C for 6 h to anneal each strand. A polynucleotide kinase (TAKARA, Japan) was used to label the 5′-end of various DNAs (10 nM) with [γ–³²P]ATP (30 nM). Labeled DNA was purified by phenol/ethanol precipitation. TmMutS2 (0, 0.5, 1.0, and 2.0 µM) was incubated with unlabeled and labeled DNAs (1 µM) at 37°C 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 µM). 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 37°C to verify conformational discrepancies among the TmMutS2 complexes. The protease-to-TmMutS2 ratio was optimized to 1:1000 (w/w) for time-resolved digestion with 5 µg of initial TmMutS2. To observe the digestion patterns of TmMutS2, 0.3 mM ADP, 1 mM ADPnP, and 1 µM 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 µL of each reaction mixture was combined with 4 µL of protein-staining buffer, followed by boiling and analysis by 15% SDS-PAGE (Figure 1D).

Size exclusion Chromatographic Analysis

TmMutS2 (10 µM) was pre-incubated with two types of nucleotides, 1 mM ADP and 1 mM ADPnP at 37°C 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 20°C to validate the conformational transitions of the TmMutS2 complexes. Eluted proteins were characterized in terms of K_av value: K_av = (V_e–V_o)/(V_t–V_o), where V_e is the elution volume of each protein, V_o is the void volume of the column, and V_t 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 endonuclease 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 µM, approximately 3 kbp size) was incubated separately with 2 µM of TmMutS2, TmMutS2ΔSmr, 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 20°C to 80°C. In addition, various metal ions were added to the TmMutS2 DNA endonucleolytic reactions. The reaction mixtures were incubated at 60°C for 2 h to help establish optimized conditions. Additionally, TmMutS2 was incubated with nucleotides at various concentrations to verify the effect of the nucleotides (Figure 2).

2 µM of TmMutL was incubated with 2 µM 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 37°C for 12 h. Each culture was diluted with 150 mL of LB medium, and then shaken at 37°C for 6 h. These cultures were spread on two types of LB plates, rifampicin-free plates or rifampicin plates containing 50 µg/mL rifampicin. The plates were incubated at 37°C 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 µM M13 phage single-stranded DNA, 1 µM SSB, 10 µM 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 37°C for 10 min [53]. For initializing the DNA strand exchange reaction, 2 µM EcoRI-linearized M13 phage dsDNA (replication form) was added to the mixtures. Various amounts of TmMutS2 were applied, and the mixture was incubated at 37°C 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 µM of TmMutS2 was incubated with 1 mM of nucleotides and 0.15 mM DNA at 45°C for 30 min, respectively. In the case of B3bp-exSmr (residue 1618-1770 of B3bp), 100 µM 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 Å; photon flux, 2×10¹¹ photon/sec; distance between the XR-II+CCD detector and the sample chamber, 2149.7 mm; the detector channel, S = 4πsinθ/λ (2θ: scattering angle, λ: wavelength); the calibration standard, BSA; and the S range of data acquisition was from 0.006 to 0.23 Å^–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 R_g 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(–4p²R_g²S²/3), where the forward scattering, I(0), is proportional to the molecular weight. About 60% points were selected to calculate the R_g values because the limit value of sR_g 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 D_max and R_g values, which were different from the R_g calculated by the Guinier approximation, were estimated by the P(R) function. Additionally, R_hydro 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∝κBT/3ηD (κB, Boltzman constant; T, absolute temperature; η, 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 D_max. 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].