Characterization of Multi-Functional Properties and Conformational Analysis of MutS2 from Thermotoga maritima MSB8 Euiyoung Jeong . , Hunho Jo . , Tae Gyun Kim., Changill Ban* Department of Chemistry, Pohang University of Science and Technology, Pohang, Gyungbuk, South Korea 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. Citation: Jeong E, Jo H, Kim TG, Ban C (2012) Characterization of Multi-Functional Properties and Conformational Analysis of MutS2 from Thermotoga maritima MSB8. PLoS ONE 7(4): e34529. doi:10.1371/journal.pone.0034529 Editor: Martin G. Marinus, University of Massachusetts Medical School, United States of America Received December 1, 2011; Accepted March 1, 2012; Published April 24, 2012 Copyright: 2012 Jeong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Research Foundation of Korea [NRF-20110013431] and by KOSEF through the Center for Electro-Photo Behaviors in Advanced Molecular Systems [20110007166]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ciban@postech.ac.kr . These authors contributed equally to this work. Introduction Prokaryotic and eukaryotic MutS homologues have been widely known as central enzymes in the DNA methyl-directed mismatch repair (MMR) mechanism [1–4]. Various studies indicate that eukaryotic MutS homologues (MSHs) are associated with not only post-replication repair of mispaired lesions but also meiosis-specific DNA recombination and DNA repair mechanisms [1,5,6]. In addition, bacterial MutS homologues have been continuously investigated because of their diverse functions [7]. Particularly, bacterial MutS2 has attracted much interest because of its high sequence homology with a eukaryotic MSH4-MSH5 heterodimer [8,9] that plays an important role in meiotic crossing-over and recombination [6,10,11]. Furthermore, sequence analysis between the MutS and MutS2 homologues implies that the MutS2 protein may have potential functions that are not shared with MutS [9,12]. For example, bacterial MutS2 homologues have shown that the deletion of the Helicobacter pylori MutS2 gene in vivo results in increases of DNA recombination, indicating that the protein suppresses homologous and homeologous DNA recombination [13–15]. It has been known that bacterial MutS2 contains a hydrolytic ATPase and possesses a selective binding affinity for DNA substrates but no specificity for homoduplex or heteroduplex DNA [16–20]. In addition, it includes a small-mutS related (Smr) domain with a non-specific nicking endonuclease function [12,17– 22]. In particular, this Smr domain is spread widely throughout many species in such proteins as the BCL-3-binding protein (B3bp) in humans [23,24], the GUN1 protein in plants [25], and the YdaL and YfcN proteins in Escherichia coli [21,26]. Although the ATPase and Smr domains are present in a large variety of prokaryotes and eukaryotes, in the case of MutS2, the biological functions as well as the functional correlations between the MutS2 domains and other homologues have not been clearly elucidated. Additionally, the conformational changes of the ATPase and Smr domains caused by binding nucleotides and DNA must be clarified. Therefore, it is necessary to study the functional characteristics and structural features of MutS2 in detail to understand the valuable functions of the protein. Small-angle X-ray scattering (SAXS) is one of the effective tools for analyzing the solution structures of bio-macromolecules, and offers various pieces of information such as domain organization, intermolecular interaction, and large-scale structure. Through the analysis of the scattering pattern at very small angles using a direct X-ray beam, the conformations of the samples can be obtained on a scale between 1 nm and several hundred nanometers [27]. This technique is readily applicable to the observation of the PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34529