conformations of many biomolecules, including DNA, RNA, and proteins [28]. Hence, the structural features of partial domains in a protein can be easily and effectively confirmed using the SAXS method. In this study, we investigated the biochemical properties and solution structures of the MutS2 protein from a hyper-thermo- philic bacterium, Thermotoga maritima MSB8, which was extracted from geothermal-heated marine sediment [29]. The nicking endonuclease activity of T. maritima MutS2 (TmMutS2) was verified by various biochemical assays, and the effects of temperature, cations, DNA, and nucleotides on endonuclease activity were examined. Additionally, the relationships between TmMutS2 and T. maritima MutL (TmMutL) or T. maritima RecA (TmRecA) were demonstrated using both in vitro and in vivo assays. In addition, the conformational transitions of TmMutS2 in the presence of nucleotides and DNA were confirmed using biochemical and SAXS modeling methods. Superimpositions of the TmMutS2 SAXS models onto the crystal structures of B3bp- Smr and the ATPase domain of Thermus aquaticus MutS (TaqMutS) indicated a resemblance between the SAXS models and the crystal structures. Results 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 identifica- tion 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 60uC compared to 37uC, and a fluorometric analysis also indicated a two-fold increase of activity at 60uC compared with 37uC (Figure S2). To clarify the effect of instability of TmMutS2, dynamic light scattering (DLS) measurements at both 37uC and 60uC 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 Functional and Conformational Analysis of TmMutS2 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 sedimenta- tion coefficient was obtained using the C(s) method [30,31]. A single narrow peak with a mean average sedimentation coefficient of 5.2 (60.2)610–13 s indicated that TmMutS2 exists as a homogeneous dimer (Figure 1B). 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 a-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 endonu- PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34529