Characterization of Multi-Functional Properties and Conformational Analysis of MutS2 from Thermotoga maritima MSB8

Abstract

The MutS2 homologues have received attention because of their unusual activities that differ from those of MutS. In this work, we report on the functional characteristics and conformational diversities of Thermotoga maritima MutS2 (TmMutS2). Various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (SPM), ATPase assays, analytical ultracentrifugation, DNA binding assays, size chromatography, and limited proteolytic analysis. Dimeric TmMutS2 showed the temperature-dependent ATPase activity. The non-specific nicking endonuclease activities of TmMutS2 were inactivated in the presence of nonhydrolytic ATP (ADPnP) and enhanced by the addition of TmMutL. In addition, TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction in a TmMutL-dependent manner. We also demonstrated that small-angle X-ray scattering (SAXS) analysis of dimeric TmMutS2 exhibited nucleotide- and DNA-dependent conformational transitions. Particularly, TmMutS2-ADPnP showed the most compressed form rather than apo-TmMutS2 and the TmMutS2-ADP complex, in accordance with the results of biochemical assays. In the case of the DNA-binding complexes, the stretched conformation appeared in the TmMutS2-four-way junction (FWJ)-DNA complex. Convergences of biochemical- and SAXS analysis provided abundant information for TmMutS2 and clarified ambiguous experimental results.

The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti‐recombination function of Helicobacter pylori MutS2

“…D and E). Contrary to other Smr domains (Fukui et al ., ; Jeong et al ., ; Zhang et al ., ), no enhancement of nuclease activity was observed due to the presence of additional amino acids at the N‐terminus of HpSmr domain (Fig. E).…”

“…In a previous study, it was reported that a dimer of Taq MutS1 (90.6 kDa) showed mean R H value of ∼ 6 nm (Biswas et al ., ). Moreover, a homogenous dimer of TmMutS2 (monomer MW 89 kDa), validated using scanning probe microscopy and analytical ultracentrifuge, showed an averaged R H value of 8.298 (± 2.05) nm at 25°C upon DLS analysis (Jeong et al ., ). The R H value for MutS2 (monomer MW 87.8 kDa) therefore suggests that its most prominent oligomeric form in solution could be tetramer.…”

“…Previous studies have suggested that MutS2 proteins may form oligomeric complexes in solution. Gel filtration chromatographic analysis of T. thermophilus MutS2 (TtMutS2) suggested that it exists mostly as higher oligomers with a small fraction as a dimer (Fukui et al, 2004), while T. maritima MutS2 (TmMutS2) occurs mostly as dimer (Jeong et al, 2012a). On the other hand, Pyrococcus furiosus MutS2 probably exists as a tetramer in solution (Vijayvargia and Biswas, 2002).…”

“…It is interesting to note that isolated TtSmr and TmSmr show elevated nuclease activity compared with the corresponding full-length MutS2 proteins (Fukui et al, 2007a;Jeong et al, 2012b). Studies with TtSmr, Deinococcus radiodurans Smr (DrSmr) and human B3BP-Smr suggested that amino acid residues adjacent to the N-terminus of Smr might regulate the nuclease activity (Fukui et al, 2007a;Jeong et al, 2012a;Zhang et al, 2014). Therefore, to verify the role of Smr domain in the nuclease activity of HpMutS2, different deletion constructs of HpMutS2 displayed in Fig.…”

Molecular basis for the functions of a bacterial MutS2 in DNA repair and recombination

“…Studies in the past decade revealed the functions of MutS2 in DNA recombination and repair. MutS2 in H. pylori as well as in some other bacteria was shown to have anti-recombination activity, playing a role in maintaining genome integrity by suppressing homologous and homeologous DNA recombination [13, 16–18]. However, B. subtilis MutS2 was recently shown to function in promoting homologous recombination [19].…”