Fluorescent Tags Can Impair DNA Mismatch Repair Figure 1. Dye tags influence single expression of MLH1 and PMS2. To determine the influence of fluorescent tags on single expressed MLH1 or PMS2 variants, HEK293T cells were transfected with different (A) MLH1 or (B) PMS2 constructs. Amounts of expressed proteins were assessed after Western blotting by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean 6S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: MLH1 unlabeled; 2: MLH1-GFP-N; 3: MLH1-GFP-C; 4: MLH1-Red-N; 5: MLH1-Red-C; 6: PMS2 unlabeled; 7: PMS2-GFP-N; 8: PMS2-GFP-C; 9: PMS2-Red-N; 10: PMS2-Red-C. doi:10.1371/journal.pone.0031863.g001 MutLa expression is influenced by fluorescent labeling The influence of dye labeling on MutLa expression rate was analyzed by quantification of MLH1 and PMS2 levels 48 h after transiently cotransfection of different variants. As shown in Figure 2, expression of fluorescent tagged-MutLa variants (Figure 2, lane 3–18) significantly differs from the untagged MutLa control (Figure 2, lane 2). MLH1 expression in all single PMS2 labeled MutLas (Figure 2, lane 3–6), in single MLH1 tagged (MLH1-GFP-N (Figure 2, lane 7) or MLH1-GFP-C (Figure 2, lane 8)) as well as in the double dye labeled MutLa variants, MLH1-GFP-C/PMS2-Red-C (Figure 2, lane 14) or MLH1-Red-C/PMS2-GFP-N (Figure 2, lane 17), was around 40–60% decreased compared to the untagged control. In contrast, expression of MLH1-GFP-N coexpressed with different Red labeled PMS2 (Figure 2, lane 11+12) was significantly higher in comparison to the control. MLH1 expression of all other MutLa variants was about 70– 80% compared to the control. Looking at PMS2 expression, completely different results were detectable. Irrespective of whether MLH1 was tagged or not, C- terminal labeling of PMS2 with Red (PMS2-Red-C (Figure 2, lane 6, 12, 14)) led to dramatically decreased expression levels of around only 10% compared to untagged PMS2. However, .100% of wild-type PMS2 expression was detectable when unlabeled PMS2 was coexpressed with MLH1-GFP-C (Figure 2, lane 8) or N-terminal labeled PMS2-GFP-N was coexpressed with MLH1-Red-N (Figure 2, lane 15). In all other tested MutLa variants PMS2 expression was on average 30–60% compared to unlabeled PMS2. Subcellular localization of dye tagged MutLa In order to analyze the influence of fluorescent proteins (GFP as well as Red) on subcellular protein localization, single expressed MLH1 or PMS2 as well as different coexpressed MutLa variants were analyzed in comparison to unlabeled MutLa using confocal laser microscopy. As shown in Figure 3, dye tagged single transfected MLH1 was, regardless of the orientation of the fluorescent tag, exclusively localized in the nucleus. In contrast PMS2-GFP-N, PMS2-Red-C as well as PMS2-Red- N were, if transfected without heterodimeric partner protein MLH1, only detectable in the cytoplasm of transfected cells. However, single transfection of the PMS2-GFP-C variant led to strong nuclear localization of PMS2 in HEK293T cells. All cotransfected MutLa variants were detected most domi- nantly in the nucleus (data not shown). MMR function is significantly impaired by fluorescent tags MMR function of Lynch syndrome variants is commonly tested by in vitro MMR-assays [16,28,29]. Beside untagged, also dye- labeled proteins are used to analyze not only MMR functionality PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31863