Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutSα is abolished by a single mutation in the hMSH6 subunit
Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutSα is abolished by a single mutation in the hMSH6 subunit
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DOI：
10.1074/jbc.m005987200
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发表时间：
2000-11-24
影响因子：
4.8
通讯作者：
Jiricny, J
中科院分区：
文献类型：
--
作者：
Dufner, P;Marra, G;Jiricny, J
人类细胞中最丰富的错配结合因子hMutS α是细菌MutS蛋白的两种同源物hMSH 2和hMSH 6的异源二聚体。所有MutS同源物的C-末端部分含有ATP结合基序，并且在整个进化过程中高度保守。虽然N末端通常是不同的，但它们也含有短的保守序列元件。在一个这样的基序GXFY（X）（5）DA内的苯丙氨酸->丙氨酸取代已经显示消除水生栖热菌MutS蛋白的错配结合活性（Malkov，V.A.，Biswas，I.，Camerini-Obery，R，D.，和Hsieh，P，（1997）J.Biol.Chem.272，23811-23817）;我们将相同的突变引入hMutS α的一个或两个亚基中。hMSH 2中的Phe → Ala取代对异二聚体的生物活性没有影响。相反，当hMSH 6亚基突变时，hMutS α的体外错配结合和错配修复功能严重减弱。此外，这种变体异源二聚体也显示出一般的DNA结合缺陷。相应地，其ATP酶活性不能被异源双链或同源双链DNA刺激。因此，hMSH 6的N-末端部分似乎赋予hMutS α：不仅识别和结合错配底物的特异性，而且结合同源双链DNA的能力。
The most abundant mismatch binding factor in human cells, hMutS alpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein. The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5)DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A, Biswas, I., Camerini-Otero, R, D., and Hsieh, P, (1997) J. Biol. Chem. 272, 23811-23817); We introduced an identical mutation into one or both subunits of hMutS alpha The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutS alpha were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA Thus the N-terminal portion of hMSH6 appears to impart on hMutS alpha: not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA.