Functional and Conformational Analysis of TmMutS2 Figure 6. The best fitted ab initio SAXS models. They are depicted with space-filled dummy atoms. doi:10.1371/journal.pone.0034529.g006 reported for the MutS-mismatched DNA complex that presented defective proteolysis with trypsin rather than apo-MutS [40]. Thus, these results imply that the TmMutS2-nucleotide complexes may have a more compact structure than apo-TmMutS2 because of their flexibility, which coincides with the result of the SEC experiment (Figure 1E). In addition, N-terminal amino acid sequencing analysis on digested TmMutS2 revealed that the largest fragment was the 118–757 residues and that the digested fragment from the TmMutS2 N-terminal was near 14 kDa. This implies that the N-terminal of the conserved DNA binding domain is not mainly engaged in direct DNA binding, having the high flexibility. Biochemical substantiations of the flexibility of the N- terminal coincide exactly with SAXS models of TmMutS2-DNA complexes, which will be debated in discussion for SAXS results. While dimerization is a common feature of many endonucle- ases, including restriction enzymes and Holliday junction-related enzymes [41,42], monomeric endonucleases also exist and are similar in size to E. coli MutH [43]. The TmMutS2-Smr domain used in this study was composed of 130 amino acid residues. Interestingly, whereas TmMutS2 had a dimeric structure (Figure 1A, 1B, and 1E), TmMutS2-Smr with high homology to T. thermophilus MutS2-Smr [18] was a monomer with no multimeric form as represented in Figure S9. The experimentally obtained molecular weight (approximately 18 kDa) of TmMutS2- Smr was comparable to the calculated molecular weight (15 kDa) of monomeric TmMutS2-Smr. Such monomeric TmMutS2-Smr shows the higher nicking endonuclease activity rather than dimeric apo-TmMutS2 (Figure 2B). Although dimerization results in a decrease of the endonuclease activity, specificity of the protein for substrates can be relatively enhanced. Therefore, it is expected that the dimeric conformation of TmMutS2 is the key feature for controlling the activity as well as specificity. MutL, one of the major proteins associated with the MMR event, interacts with MutS [1–4,44]. It also stimulates the nicking endonuclease activity of MutH in the E. coli MMR system [1,3,4], in which the C-terminal region of MutL plays an important role in the activation of MutH [45]. Importantly, this implies that a protein having a relative sequence homology with the C-terminal regions of MutL in the bacterial systems lacking MutH [17] may have a complementary interaction with MutL. Thus, there is a possibility that the C-terminal regions of MutL interact with the Smr domain of MutS2. Non-specific nicking endonuclease activity of TmMutS2, that is dependent on diverse factors such as temperature, amount and type of cations, and the types of nucleotides, increased following the addition of TmMutL (Figure 3A). These results suggest that interactions between TmMutS2 and TmMutL successfully improve the nicking endonuclease activity of TmMutS2. However, ADPnP negatively affected the nicking endonuclease activity of TmMutS2, irrespec- tive of the presence of TmMutL (Figure 2C, 3B), indicating that the ATPase domain is close to the Smr domain and far from the binding site of TmMutL. This proximity will be further discussed in the SAXS part. In the various eukaryotic DNA repair mechanisms, nicking endonucleases are involved in the excision of damaged DNA as well as the recognition of specific nicked sites in strand displacement reactions [46]. Recent results demonstrate that the bacterial and eukaryotic MutS2 sequence homologues have the ability to identify damaged DNAs and bind to it [6,15,17]. The sequence alignment of MutS2 homologues suggests that the Smr domain of MutS2 can introduce a nick in DNA, and then the nick is extended by other exonucleases, resulting in undesirable strand exchanges and the early termination of recombination. Therefore, the mutation frequency can be influenced by MutS2 due to its endonuclease activity. In practice, the incorporation of the exogenous the TmmutS2 gene into the E. coli chromosome containing RecA yielded the important result that TmMutS2 inhibited the RecA-mediated recombination reaction in vivo and in vitro (Figure 4). Interestingly, the DNA strand-exchange reaction was further negatively impacted by the addition of TmMutL, which provided the surprising clue that TmMutS2-TmMutL interaction might be an essential or additional condition for DNA homologous recombination (Figure 4C) similar to the MutS-MutL complex [33]. In the human recombination system, the MSH4- MSH5 heterodimer interacts with the MutLa heterodimer that has an endonuclease function (MLH1-PMS2 in human and MLH1-PMS1 in yeast) [47]. However, sequence analysis of TmMutS2 indicates that the protein lacks any known nuclease motif present in the MutLa heterodimer. Although TmMutS2 shows an unlinked relationship by primary sequence analysis, the PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34529