Mutation in the magnesium binding site of hMSH6 disables the hMutSα sliding clamp from translocating along DNA
Mutation in the magnesium binding site of hMSH6 disables the hMutSα sliding clamp from translocating along DNA
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DOI：
10.1074/jbc.275.3.2080
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发表时间：
2000-01-21
影响因子：
4.8
通讯作者：
Jiricny, J
中科院分区：
文献类型：
--
作者：
Iaccarino, I;Marra, G;Jiricny, J
在人类细胞中，碱基/碱基错配和小插入/缺失环的结合是由hMutSα介导的，hMutSα是hMSH2和hMSH6的异源二聚体，在ATP和镁的存在下，hMutSα通过以滑动钳的形式沿着DNA轮廓解离错配。这一过程是由异二聚体的构象变化实现的，而异二聚体的构象变化是由多肽的A型和B型Walker基序中的ATP和镁的结合驱动的。我们发现，一个纯化的重组hMutSα变异体hMutSα6DV在hMSH6亚基的B型Walker基序中含有天冬氨酸到Valine的取代，不能经历与反式定位相容的构象变化。相反，在ATP和镁的存在下，它与含有错配的DNA底物直接解离，从而排除了功能错配修复复合体的组装。野生型hMutSα的“易位”构象仅在体外有利于核苷酸水解和错配修复的条件下才能观察到。因此，虽然镁可以被锰取代，但三磷酸腺苷不能分别被其缓慢或不可水解的同系物S或AMPPNP所取代。ATP在有镁和无镁的情况下诱导hMutSα发生不同的构象变化，这一发现有助于解释不能结合ATP的hMutSα变体与不能结合金属离子的hMutSα变体之间的功能差异。
In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutS alpha, a heterodimer of hMSH2 and hMSH6, In the presence of ATP and magnesium, hMutS alpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutS alpha variant, hMuts alpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with trans location. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The "translocation-prone" conformation of wild type hMutS alpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gamma S or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutS alpha in the presence and in the absence of magnesium helps explain the functional differences between hMutS alpha variants incapable of binding ATP as compared with those unable to bind the metal ion.