Habraken Y, Sung P, Prakash L, Prakash S
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061, USA.
J Biol Chem. 1998 Apr 17;273(16):9837-41. doi: 10.1074/jbc.273.16.9837.
MSH2 and MSH6 proteins exist as a stable complex, as do the MLH1 and PMS1 proteins. To study the mismatch binding properties of the MSH2-MSH6 complex and to examine its functional interaction with the MLH1-PMS1 complex, these protein complexes were purified to near homogeneity from overproducing yeast strains. As has been reported previously, the purified MSH2-MSH6 complex binds DNA substrates containing a G/T mismatch and insertion/deletion mismatches, but the binding affinity for the latter decreases as the size of the extrahelical loop increases. Addition of ATP or the nonhydrolyzable ATPgammaS reduces binding of the MSH2-MSH6 complex to the DNA substrates markedly. Here, we show that MSH2-MSH6 forms a ternary complex with MLH1-PMS1 on a mismatch containing DNA substrate. The formation of this ternary complex requires ATP, which can be substituted by ATPgammaS, suggesting that ATP binding alone is sufficient for ternary complex formation. Thus, it appears that ATP binding by the MSH2-MSH6 complex induces a conformation that is conducive for the interaction with MLH1-PMS1 complex, leading to the formation of the ternary complex.
MSH2和MSH6蛋白以稳定复合物的形式存在，MLH1和PMS1蛋白也是如此。为了研究MSH2 - MSH6复合物的错配结合特性，并检测其与MLH1 - PMS1复合物的功能相互作用，从过量表达的酵母菌株中纯化这些蛋白复合物至近乎均一。如先前报道，纯化的MSH2 - MSH6复合物可结合含有G/T错配和插入/缺失错配的DNA底物，但随着螺旋外环大小增加，其对后者的结合亲和力降低。添加ATP或不可水解的ATPγS可显著降低MSH2 - MSH6复合物与DNA底物的结合。在此，我们表明MSH2 - MSH6在含有错配的DNA底物上与MLH1 - PMS1形成三元复合物。该三元复合物的形成需要ATP，ATPγS可替代ATP，这表明仅ATP结合就足以形成三元复合物。因此，似乎MSH2 - MSH6复合物结合ATP会诱导一种有利于与MLH1 - PMS1复合物相互作用的构象，从而导致三元复合物的形成。
