Department of Medicine I, University of Frankfurt/M., Frankfurt, Germany.
PLoS One. 2012;7(2):e31863. doi: 10.1371/journal.pone.0031863. Epub 2012 Feb 14.
The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
人类 DNA 错配修复（MMR）过程对于维持基因组的完整性至关重要，需要许多不同的蛋白质相互完美协调。MMR 基因的种系突变导致称为林奇综合征的遗传性结直肠癌的发生。到目前为止，已经鉴定出各种突变主要在两种 MMR 蛋白 MLH1 和 MSH2 中，而 55%在 MLH1 中检测到，MLH1 是异二聚体 MutLα（MLH1 和 PMS2）的必需成分。大多数 MLH1 变体是致病性的，但错义突变的相关性常常不清楚。应用了许多不同的重组系统来筛选出与疾病相关的蛋白质，其中经常使用荧光标记的蛋白质。然而，染料标记可能对 MutLα 的功能产生有害影响。因此，我们分析了 N 端和 C 端荧光标记对 MutLα 的表达水平、细胞定位和 MMR 活性的影响。除了 GFP-或 Red-融合对蛋白质表达的显著影响外，我们还检测到单个表达的 C 端 GFP 标记的 PMS2 错误穿梭到细胞核中，并发现 C 端染料标记会损害 MutLα 的 MMR 功能。相比之下，C 端标记的 MutLα 保留了正确的功能，既可以用于细胞定位分析，也可以用于 MMR 效率分析。
