Bjornson Keith P, Blackwell Leonard J, Sage Harvey, Baitinger Celia, Allen Dwayne, Modrich Paul
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2003 Sep 5;278(36):34667-73. doi: 10.1074/jbc.M305513200. Epub 2003 Jun 25.
Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 x 10(7) M-1, indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703-710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717). MutS800 binds a 20-base pair heteroduplex an order of magnitude more weakly than full-length MutS, and at saturating protein concentrations, the heteroduplex-bound mass observed with MutS800 is only half that observed with the full length protein, indicating that the subunit copy number of heteroduplex-bound MutS is twice that of MutS800. Analytical equilibrium ultracentrifugation using a fluorescein-tagged 20-base pair heteroduplex demonstrated that native MutS forms a tetramer on this single site-sized heteroduplex DNA. Equilibrium fluorescence experiments indicated that dimer-to-tetramer assembly promotes mismatch binding by MutS and that the tetramer can bind only a single heteroduplex molecule, implying nonequivalence of the two dimers within the tetramer. Compared with native MutS, the ability of MutS800 to promote MutL-dependent activation of MutH is substantially reduced.
分析型平衡超速离心表明，大肠杆菌MutS以二聚体和四聚体的平衡混合物形式存在。二聚体向四聚体转变的缔合常数为2.1×10⁷ M⁻¹，这表明在生理浓度下该蛋白质将同时包含二聚体和四聚体。MutS的羧基末端是四聚体组装所必需的，因为先前描述的53个氨基酸的羧基末端截短体（MutS800）形成了一种有限的二聚体物种（Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703 - 710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711 - 717）。MutS800与20碱基对异源双链体的结合比全长MutS弱一个数量级，并且在蛋白质浓度饱和时，MutS800观察到的异源双链体结合质量仅为全长蛋白质观察到的一半，这表明异源双链体结合的MutS的亚基拷贝数是MutS800的两倍。使用荧光素标记的20碱基对异源双链体进行的分析型平衡超速离心表明，天然MutS在这种单位点大小的异源双链体DNA上形成四聚体。平衡荧光实验表明，二聚体到四聚体的组装促进了MutS与错配的结合，并且四聚体只能结合单个异源双链体分子，这意味着四聚体内的两个二聚体是不等价的。与天然MutS相比，MutS800促进MutL依赖的MutH激活的能力大幅降低。
