Steady-state regulation of the human DNA mismatch repair system
Steady-state regulation of the human DNA mismatch repair system
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DOI：
10.1074/jbc.m001140200
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发表时间：
2000-06-16
影响因子：
4.8
通讯作者：
Boland, CR
中科院分区：
文献类型：
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作者：
Chang, DK;Ricciardiello, L;Boland, CR
用新发展的竞争性定量逆转录聚合酶链式反应和用纯化的蛋白质归一化的Western分析，检测了人类DNA错配修复(MMR)转录本和蛋白质在MMR熟练和缺陷细胞系中的稳态水平。在精通MMR的细胞中，hMSH2是最丰富的MMR蛋白，其表达水平是hMLH1的3-5倍。HMLH1蛋白的表达是hPMS2的1.5~2.5倍。HMSH2突变的LoVo细胞不表达可检测到的hMSH3或hMSH6蛋白，hMLH1突变的HCT116细胞不表达可检测到的hMLH1或hPMS2蛋白，而hMLH1恢复的HCT116+CH3细胞可重新表达hPMS2蛋白。在hMSH6突变的HCT15细胞中，hMSH3蛋白和信使核糖核酸均增加。在SV40转化的肺成纤维细胞中，所有被检测的MMR mRNAs和蛋白的表达水平都比未转化的肺成纤维细胞高1.5-5倍。MMR蛋白的稳定水平表明，与hMutL蛋白相比，参与DNA错配识别的hMutS蛋白存在更多的hMutS蛋白。HMSH3和hMSH6蛋白的稳定性似乎取决于hMSH2蛋白的存在，类似地，hPMS2蛋白的稳定性取决于hMLH1。当hMSH6突变失活时，hMSH3通过转录上调和蛋白质稳定性增强而增加。在成纤维细胞模型中，病毒转化后所有成分的平衡上调。MMR组分的定量变化是改变细胞DNA MMR能力的潜在机制。
Steady-state levels of human DNA mismatch repair (MMR) transcripts and proteins were measured in MMR-proficient and -deficient cell lines by the newly developed competitive quantitative reverse transcription- polymerase chain reaction and Western analysis normalized with purified proteins. In MMR-proficient cells, hMSH2 is the most abundant MMR protein and is expressed 3 to 5 times more than hMLH1. The hMLH1 protein was expressed 1.5 to 2.5 times more than hPMS2. Steady-state levels of mRNA expression correlated well with protein expression, hMSH2-mutated LoVo cells did not express detectable hMSH3 or hMSH6 proteins, Similarly, hMLH1-mutated HCT116 cells did not express detectable hMLH1 or hPMS2 protein, whereas in hMLH1-restored HCT116+ch3 cells, hPMS2 protein was reexpressed. In hMSH6-mutated HCT15 cells, both hMSH3 protein and mRNA were Increased. In SV40-transformed lung fibroblasts, all MMR mRNAs and proteins examined were expressed at levels 1.5-5-fold higher than in their nontransformed counterpart. The steady-state levels of MMR proteins indicate that substantially more hMutS proteins, which are involved in DNA mismatch recognition, are present in comparison with the hMutL proteins. Stability of hMSH3 and hMSH6 proteins appears to depend upon the presence of the hMSH2 protein, and, similarly, the stability of the hPMS2 protein depends upon hMLH1. When the hMSH6 is mutationally inactivated, hMSH3 increases by both transcriptional up-regulation and enhanced protein stability. A balanced up-regulation of all of the components was seen after viral transformation in a fibroblast model. Quantitative changes of the MMR components are a potential mechanism to modify the DNA MMR capabilities of a cell.