Figure 3. Subcellular localization of single expressed MLH1 and PMS2 variants. HEK293T cells were transfected with different MLH1 or PMS2 constructs as indicated to the left. MLH1 and PMS2 were visualized after 48 h using confocal laser microscopy. Nuclei were counterstained with TO-PRO-3 (middle column) after fixation and resulting overlay is shown in the right column. As a control, unlabeled MLH1 or PMS2 was transfected in parallel and visualized after 48 h using FITC-labeled goat anti-mouse IgG Alexa Fluor 488 for MLH1 detection (shown in green; left column) whereas unlabeled PMS2 was detected with the FITC-labeled goat anti-rabbit IgG Alexa Fluor 555. All used labeled MLH1 and PMS2 constructs showed the same cellular localization as the unlabeled form, with the exception of PMS2-GFP-C which was detected in the nucleus although all other constructs were located in the cytoplasm. doi:10.1371/journal.pone.0031863.g003 Fluorescent Tags Can Impair DNA Mismatch Repair but also the influence of mutations on cellular localization and interaction in parallel [10,17]. Hereby, several different MMR- fusion-proteins were utilized carrying e.g. either N- or C-terminal dye tags. A dye-dependent change of protein function cannot be excluded especially for C-terminal protein labeling since the very sensitive MLH1/PMS2 interaction zone as well as the endonuclease function are located at the C-terminus and of great importance for the MMR function of MutLa [28]. Therefore, we determined the influence of the fluorescent tag orientation on the MMR functionality of MutLa. All single PMS2 tagged (N- or C-terminal) MutLas were still MMR proficient showing 80% or more MMR activity (Figure 4, lane 3–6). In contrast, most single MLH1 labeled (with the exception of MLH1-GFP-N (Figure 4, lane 7) and most double fluorescent labeled MutLa-variants showed less than 50% MMR activity compared to wild-type MutLa. Hereby, MLH1-GFP-C coupled with PMS2-Red-C or PMS2-Red-N, respectively showed the most limited functionality. C-terminal GFP or N- and C-terminal Red MLH1 labeling with or without combination of fluorescent labeled PMS2 showed only #50% repair activity thus were MMR deficient. In contrast, dye tagged MutLa, consisting of N-terminal tagged MLH1 and N-terminal fused PMS2 showed most MMR functionality whereby MLH1-GFP-N/PMS2-Red-N showed 70– 85% MMR activity and therefore seems to be the most useful dye tagged MutLa variant. Discussion Functional testing of MMR protein variants is of great importance to distinguish pathogenetic relevant mutations and non-pathogenetic polymorphisms detected in Lynch syndrome families [16,30,31]. Beside repair activity, also cellular localization and protein stability serve for the classification and dye tagged variants are frequently used to simplify MMR-protein analysis [2,8,16,30]. However, a negative impact of fluorescent labeling on proteins’ functionality might be present [18,19,20]. Therefore, we compared expression level, cellular localization and MMR activity of untagged and dye tagged MutLa variants and found significant changes in all parameters. Expression levels of fluorescent labeled MutLas significantly differ from unlabeled MutLa. As shown by our results, fusion of GFP and Red can dramatically affect protein stabilization. N- terminal fusion of dyes e.g. lead to stabilization even of single expressed PMS2 which is normally unstable without its heterodi- meric partner. Our observation of this dye-dependent effect is in accordance to previously published data describing the large list of possible effects using fluorescent fusion proteins [32]. Thus, fluorescent tagged proteins do not seem to be useful for protein stability analysis. However, differences in protein expression seemed to be without significant impact on MMR functionality of the heterodimer. Weak protein-expression, e.g. of tagged PMS2, did not automatically cause dramatic MMR decrease or vice versa, which is in accordance to Cejka et al. who detected that the amount of overexpressed MutLa is not the limiting factor in the MMR assay so that protein expression could be strongly reduced without functional impact [33]. Looking at the influence of dye labeling on MMR in detail, dye tags generally decrease repair function of MutLa even if only one partner protein was labeled. Hereby, labeling of MLH1 had a stronger restrictive effect than PMS2 tagging which illustrates the PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31863