Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D
Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D
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DOI：
10.1074/jbc.m111.316919
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发表时间：
2012-03-23
影响因子：
4.8
通讯作者：
Hsieh, Peggy
中科院分区：
文献类型：
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作者：
Geng, Hui;Sakato, Miho;Hsieh, Peggy
异二聚体人MSH 2-MSH 6蛋白通过识别由复制错误引起的错配碱基来启动DNA错配修复（MMR）。在MSH 2的ATP结合位点或MSH 6的ATP水解催化位点中或附近具有突变的Msh 2（G674 A）或Msh 6（T1217 D）小鼠发生癌症，并且由于MMR途径的丧失而具有缩短的寿命（Lin，D. P.的一种，王玉，谢勒，S. J.，克拉克，A. B.，杨，K.，Avdievich，E.，Jin，B.，沃林大学，Parris，T.，Kurihara，N.，奥马尔，A.，库切拉帕蒂河，Lipkin，M.，Kunkel，T.一、Edelmann，W.（2004）Cancer Res.64，517522; Yang，G.，谢勒，S. J.，谢尔，S。美国，杨，K.，金，M.，Lipkin，M.，库切拉帕蒂河，科洛德纳河D、Edelmann，W.（2004）Cancer Cell 6，139150）。来自这些小鼠的小鼠胚胎成纤维细胞保留对DNA损伤的凋亡反应。在多种功能测定中对突变的人MutS α蛋白MSH 2（G674 A）-MSH 6（wt）和MSH 2（wt）-MSH 6 T（1219 D）进行了分析，并且如预期的那样不能支持体外MMR，尽管它们保留了错配识别活性。DNA结合和ATP酶活性的动力学分析和MMR的切除步骤的检查表明，这两个突变体在其潜在的分子缺陷不同。MSH 2（wt）-MSH 6（T1219 D）不能偶联核苷酸结合和错配识别，而MSH 2（G674 A）-MSH 6（wt）在核苷酸结合中具有部分缺陷。然而，这两种突变蛋白仍然与错配结合，并且不能促进有效切除，从而以显性方式在体外抑制MMR。这些研究结果的MMR和DNA损伤信号的MMR蛋白的影响进行了讨论。
The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutS alpha proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6T(1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.