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. doi:10.1371/journal.pone.0034529.g007 biological functions of eukaryotic MutS2 sequence homologues in DNA recombination system may be analogous to the DNA binding and endonucleolytic activities of TmMutS2. In addition to the biochemical characterization of TmMutS2, SAXS offered structural information on the protein and made it possible inspect the structural features of TmMutS complexes with nucleotides and DNA. Through the modeling of the various complexes, the TmMutS2-ADP model was confirmed to have a compacted form contrary to the extended structures of TmMutS2- DNAs models and in accordance with biochemical results (Figure 6). Therefore, it is expected that this structural compact- ness has the possibility of suppressing the nicking endonuclease activity of TmMutS2. In the case of the superimposition of the ATPase domain (residue 310–502) to the SAXS models, the domain was localized in the center of TmMutS2. Additionally, the superimpositions among the TmMutS2-ADPnP SAXS model and the TaqMutS- ATPase structure/TmMutS2-ATPase model indicate that dimer interfaces maybe exist in the middle region (Figure 7B). Functional and Conformational Analysis of TmMutS2 The TmMutS2-FWJ-DNA SAXS model had a large central cavity in the superimposed region and retained a more globular form compared to the other SAXS models (Figure 8B). Also, superimposition of TmMutS2-FWJ-DNA or -dsDNA SAXS models to the B3bp-Smr structure or the TmMutS2-Smr model exhibits an intrinsically flexible N-terminal that would allow for a certain conformational variability in the apo-TmMutS2 and TmMutS2-nucleotides SAXS models (Figure 6). Particularly, TmMutS2-FWJ-DNA represents the most elongated form, implying that dimeric interactions of the protein enhance the binding affinity for DNA substrates. This result certainly agrees with the biochemical experiments as previously mentioned. Therefore, it is clear that the N-terminal domain of TmMutS2 plays an important role in efficient binding for various types of DNA via conformational transitions. Additionally, the superim- position of the TmMutS2-DNA model to the B3bp-exSmr SAXS model reveals that TmMutS2 retains the structurally conserved Smr domain in the C-terminal and the DNA-binding region in the large central cavity (Figure 8C). This feature is in accordance with the result of previous biochemical assay that ADPnP inhibits the nicking endonuclease activity of TmMutS2. Since both the ATPase and the Smr domains are located in the center of TmMutS2, ADPnP binding interrupts the endonuclease activity of the Smr domain. Diverse MutS homologues recognize specific DNA substrates such as mismatched DNAs, bulged loops, oxidative-modified bases, and FWJ-DNAs. Besides these DNA-protein interactions, endonucleolytic activity is also required for DNA repair mecha- nisms [45]. TmMutS2 exhibited non-specific DNA-binding activity and nicking endonuclease activity. In addition, TmMutS2 interacted with TmMutL and regulated the RecA-mediated DNA exchange reaction similar to the MutS2 of other species [15,17,20]. Therefore, bacterial MutS2 including TmMutS2 may bind to random DNA, particularly cross-over DNA, and affect recombination reactions. Additionally, the results from our investigations demonstrate that various events related to TmMutS2 and the structure of TmMutS2 can be affected by binding with nucleotides and DNA. The SAXS modeling method was very useful for inspecting the other intermediate conforma- tions of the protein complexes and for further demonstrations of the structure of the partial domains such as ATPase and DNA- binding domains. This amalgamation of biochemical- and SAXS analysis offered valuable information for conformational transi- tions of TmMutS2 and substantiated various experimental results. At present, to explore the structural and biochemical charac- teristics of TmMutS2 in more detail, our group has been continuously trying to determine the high-resolution structure of TmMutS2 using both X-ray crystallographic and the SAXS-based methods. The experimental findings described in this study may help to understand the multiple functions and structural characteristics of TmMutS2. It is expected that these investigations will provide critical insights into the functionalities of the prokaryotic and eukaryotic MutS2 sequence homologues involved in DNA homologous recombination, as well as aid in perfecting comprehension of the accurate and detailed functions of MutS2. Materials and Methods 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 comple- mentary DNA. All genes were amplified by PCR using specific primers as shown in Table S2. The amplified genes were inserted PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34529