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

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

Highly sensitive and selective in vitro diagnostics based on DNA probes and aptamers

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Enzyme-linked antibody aptamer assays based colorimetric detection of soluble fraction of activated leukocyte cell adhesion molecule

Patients with pancreatic and colorectal cancers have elevated levels of activated leukocyte cell adhesion molecule (ALCAM) in their blood compared to healthy individuals. To this end, sensitive detection of soluble fraction of ALCAM (sALCAM), also called CD166, was performed using enzyme-linked antibody aptamer (ELAA) assay. For the assay, aptamer specific for sALCAM was used as a capturing probe and polyclonal antibody modified with an enzyme was used as a detecting probe for colorimetric visualization. The ELAA assay was able to detect sALCAM as low as 0.31 ng/mL. In addition, the assay enabled the differentiation of sALCAM from other proteins in buffer as well as in human serum. The ELAA assay provides cost-effective, highly sensitive, and reproducible detection of sALCAM. (C) 2016 Elsevier B.V. All rights reserved.1111sciescopu

Nicking endonuclease activity of TmMutS2.

<p>(<b>A</b>) The effects of various cations on the nicking endonuclease activity of TmMutS2. The cations were used at a concentration of 10 mM. The nick, l, and sc indicate the nicked, linearized, and supercoiled open circular DNA forms, respectively. (<b>B</b>) A mixture containing 2 µM TmMutS2/TmMutS2-Smr and 1.5 µM plasmid DNA (linear size, 3 kbp) was incubated at temperatures ranging from 20°C to 80°C in the presence or absence of Mg²⁺ (upper plot). In the middle of the gel, M indicates the DNA size marker. The lower plot presents the percentage yields of the nicked form. (<b>C</b>) Different activities of DNA digestion in the presence of ATP (lanes 3 to 7), ADP (lanes 8 to 12), and ADPnP (lanes 13 to 17). Lane 1 and 2 are TmMutS2 untreated- and treated-plasmid DNA bands in the absence of theh nucleotide at 60°C. Each nucleotide was utilized at a final concentration of 3 mM.</p

Superimpositions among the TmMutS2-dsDNA/-FWJ-DNA SAXS models and the Smr structure/model.

<p>(<b>A</b>) The left structure is the monomeric Smr domain of <i>H. sapiens</i> B3bp (B3bp-Smr), and the right model is the predicted monomeric Smr domain of TmMutS2 (TmS2-Smr). (<b>B</b>) Superimpositions of the TmMutS2-dsDNA/-FWJ-DNA SAXS model to the B3bp-Smr structure and the TmS2-Smr model. (<b>C</b>) Superimpositions of the TmMutS2-dsDNA/-FWJ-DNA SAXS model to the B3bp-exSmr SAXS model (green mesh). The B3bp-Smr structure (red) fits well with the B3bp-exSmr SAXS model.</p

Biochemical properties of TmMutS2.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) DNA binding activities of TmMutS2 in the absence or presence of ATP (1 mM). TmMutS2 concentration is varied from 0 to 2 µM. 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 (1′–4′), in the gel. (<b>D</b>) Limited digestion by α-chymotrypsin. Lane 1 represents the protein molecular weight marker. Lane 2 is the protease-untreated TmMutS2 (5 µg). TmMutS2 was digested in the presence of 0.3 mM ADP (lanes 5–6), 1 mM ADPnP (lanes 7–8), and 1 µM 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. (<b>E</b>) 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 (V_e). The protein standards were used for the standardization of molecular weights using K_av = (V_e–V_o)/(V_c–V_o), where V_c is the geometric column volume and V_o is the void volume that is calculated by Blue dextran (2000 kDa).</p

Suppression of the TmRecA-mediated DNA recombination reaction by TmMutS2.

<p>(<b>A</b>) A spontaneous mutation frequency assay using the incorporation of TmmutS2 gene into the <i>E. coli</i> BL21(DE3) strain. Rif^R indicates rifampicin-resistance. The frequency of Rif^R (y-axis)/10⁸ cells is quantitatively analyzed by counting the number of spontaneously mutated colonies. (<b>B</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. (<b>C</b>) To observe the influence of TmMutL in the DNA strand exchange reaction, TmRecA, TmMutS2, and TmMutL were incubated at 37°C.</p