Functional and Conformational Analysis of TmMutS2 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. RifR indicates rifampicin-resistance. The frequency of RifR (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 37uC. doi:10.1371/journal.pone.0034529.g004 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 PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34529