Functional and Conformational Analysis of TmMutS2 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 mM. 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 (19–49), in the gel. (D) Limited digestion by a-chymotrypsin. Lane 1 represents the protein molecular weight marker. Lane 2 is the protease-untreated TmMutS2 (5 mg). TmMutS2 was digested in the presence of 0.3 mM ADP (lanes 5–6), 1 mM ADPnP (lanes 7–8), and 1 mM 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 (Ve). The protein standards were used for the standardization of molecular weights using Kav = (Ve–Vo)/(Vc–Vo), where Vc is the geometric column volume and Vo is the void volume that is calculated by Blue dextran (2000 kDa). doi:10.1371/journal.pone.0034529.g001 clease 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 20uC to 60uC (left three lanes). In addition, TmMutS2-Smr also exhibited temperature-dependent endonuclease activity, but activity decreased in harsh conditions such as temperatures of 80uC (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 TmMutS2DSmr were incubated with supercoiled circular dsDNA (scDNA). TmMutS2DSmr presented no nicking endo- nuclease 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. 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. 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 (60.03)610–8 and 0.91 (60.07)610–8 rifampicin-resistance colo- nies, respectively), while incorporation of the TmmutS2 gene into pET-28aTEV resulted in an approximately 6-fold increase of the spontaneous mutation frequency (6.63 (60.13)610–8). Addition- ally, the mutation on the Smr domain caused conspicuous decrease of the mutation frequency in comparison with wild type TmMutS2 (2.67 (60.20)610–8 and 7.33 (60.47)610–8 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]. 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 linear- ized 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 reac- tion. 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. PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34529