Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, Connecticut 06459, and.
J Biol Chem. 2012 Mar 23;287(13):9777-9791. doi: 10.1074/jbc.M111.316919. Epub 2012 Jan 25.
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, 517-522; 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, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) 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.
人源 MSH2-MSH6 异二聚体通过识别复制过程中产生的错配碱基来启动 DNA 错配修复（MMR）。MSH2（G674A）或 MSH6（T1217D）突变小鼠，其 MSH2 的 ATP 结合位点或 MSH6 的 ATP 水解催化位点发生突变，会由于 MMR 途径的缺失而患上癌症，并因此缩短寿命（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, 517-522; 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, 139-150）。这些小鼠的胚胎成纤维细胞对 DNA 损伤仍保留有凋亡反应。突变的人 MutSα 蛋白 MSH2（G674A）-MSH6（wt）和 MSH2（wt）-MSH6（T1219D）在各种功能测定中进行了分析，如预期的那样，它们无法在体外支持 MMR，尽管它们仍保留错配识别活性。DNA 结合和 ATP 酶活性的动力学分析以及 MMR 切除步骤的检查表明，这两种突变体在其潜在的分子缺陷方面存在差异。MSH2（wt）-MSH6（T1219D）不能偶联核苷酸结合和错配识别，而 MSH2（G674A）-MSH6（wt）在核苷酸结合方面存在部分缺陷。然而，这两种突变蛋白仍与错配结合，并不能有效地促进切除，从而以显性方式抑制体外 MMR。讨论了这些发现对 MMR 以及 MMR 蛋白的 DNA 损伤信号转导的影响。
