Fluorescent Tags Can Impair DNA Mismatch Repair Figure 4. Influence of fluorescent tags on MMR activity of MutLa. HEK293T cells were transiently cotransfected with various labeled or unlabeld MutLa constructs and 48 h post transfection MMR activity of different MutLas were assessed in vitro in parallel with unlabeled MutLa by quantifying the 39-nick-directed correction of a G-T mismatch in a restriction site of a plasmid substrate as detailed in ‘‘Materials and Methods’’ and as previously described [10]. In vitro repair was scored on a 2-kb circular DNA substrate that contains an EcoRV site which is destroyed by a G-T mismatch. Upon repair of the G-T mismatch to an A-T base pair the intact EcoRV site together with an AseI site gives rise to a 0.8- and a 1.2-kb fragment, whereas unrepaired DNA is only linearized by AseI to a 2-kb fragment. Repair efficiency was assessed by measuring the signal intensities of linearized and digested vector with Bio-Rad Quantity One software using the ‘‘rolling ball’’ baseline correction. The signal intensity of the repair bands was divided by the intensity of all three bands. Repair efficiency of unlabeled MutLa was set at 100 percent and repair of fluorescent tagged MutLa was determined in relation to the wild-type sample that was expressed, processed and tested in parallel. Average repair values and standard deviations (6) were determined from four independent experiments. Single PMS2 tagged MutLas, single MLH1-GFP-N tagged MutLas as well as MLH1-GFP-N coexpressed with PMS2-Red-N or MLH1-Red-N coexpressed with PMS2-GFP-N were MMR proficient while all other tagged variants showed MMR deficiency. 1: mock control (untransfected). 2: MLH1/PMS2 unlabeled (positive control); 3: MLH1/PMS2-GFP-N; 4: MLH1/PMS2-GFP-C; 5: MLH1/PMS2-Red-N; 6: MLH1/PMS2-Red-C; 7: MLH1-GFP-N/PMS2; 8: MLH1-GFP-C/PMS2; 9: MLH1-Red-N/PMS2; 10: MLH1-Red-C/PMS2; 11: MLH1-GFP- N/PMS2-Red-N; 12: MLH1-GFP-N/PMS2-Red-C; 13: MLH1-GFP-C/PMS2-Red-N; 14: MLH1-GFP-C/PMS2-Red-C; 15: MLH1-Red-N/PMS2-GFP-N; 16: MLH1- Red-N/PMS2-GFP-C; 17: MLH1-Red-C/PMS2-GFP-N; 18: MLH1-Red-C/PMS2-GFP-C. Symbols see Figure 1. doi:10.1371/journal.pone.0031863.g004 well known great importance of MLH1 for MutLa stability, transport and thereby MMR function [2,22,34]. Fluorescent tags fused to the C-terminus of MutLa components, most obvious GFP-fusion to the C-terminus of MLH1 entirely impair MMR function of the heterodimer. However, exclusively N- terminal tagged MutLas showed MMR proficiency which empha- sizes that the N-termini of MLH1 and PMS2 are much more tolerant to dye protein extension than their C-termini. Since MutLa was described to coordinate protein-protein interaction during the MMR process via its N-terminus [35] the observed tolerance of this domain to fluorescent dyes are explainable. In contrast, we assume that C-terminal tags might lead to incorrect interaction of the heterodimeric partners or to a strong hindrance of the MutLa radius of action which would fit well to the great importance of the last C-terminal alpha helix of MLH1 for nucleotide binding and correction of mispairs [2]. Moreover, this postulation is enforced by our computer modeling of a three-dimensional structure of dye labeled MutLa (Figure 5) consisting of previously published homology models of MLH1 and PMS2 [24,25,26,27] and structures of GFP or Red (PDB number: 2WSN (GFP); PDB number: 1G7K (Red)). C-terminal interaction of MLH1 and PMS2 fix the C- terminus of the heterodimeric complex and additional adhesion of dye complexes might hide the functional surface and lead to the observed impaired ability for MMR. Although GFP and Red show only marginal differences in structure, GFP overall had less impact on MutLa function than Red. Looking at the influence of fluorescent fusion on cellular protein localization we found one dye-dependent incorrect protein transport. While PMS2 is normally not able to enter the nucleus without dimeric partner protein MLH1 [2,11,22,34] C-terminal labeling with GFP (although PMS2-GFP-C was very weak expressed) wrongly enables this protein to be shuttled into the nucleus. This nuclear localization of single expressed C-terminal dye tagged PMS2 was previously detected by Raevaara et al. as well as Leong et al. who used a PMS2-GFP-C or a PMS2-Red-C construct, respectively [13,15]. Leong and coworkers thereupon postulated a weak nuclear import ability of PMS2. However, we presume that nuclear import of C-terminal GFP tagged single expressed PMS2 is only a dye-depending phenomenon and might lead to an incorrect interpretation of mutational influence of MLH1 on PMS2 transport mechanism. In order to overcome the described problems, one might consider switching the tags to any other of the numerous fluorescent dyes available. However, due to high structural similarities between all these dyes we do not expect relevant functional differences compared to those generated with GFP or Red. Impaired repair function of MutLa by C-terminal labeling has to be assumed using any of these fluorescent dyes. In summary, only fusion of fluorescent proteins to the N-termini of MLH1 and PMS2 enable correct functionality and cellular localization of MutLa and are recommended for testing of MutLa variants without restrictions. Materials and Methods Cells and Cell Transfection HEK293T cells, obtained from Dr. Kurt Ballmer (Paul Scherer Institute, Villingen, Switzerland) were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FCS. HEK293T is a clone of HEK293 that expresses the SV40 large T antigen. As previously published, MutLa is not expressed in HEK293T [21]. Transfection and cotransfection of HEK293T cells were carried out as described previously [7]. In brief, HEK293T were transfected at 50–70% confluence with expression plasmids (1 mg/ml, respec- tively) using 10 ml/ml of the cationic polymer polyethylenimine (Polysciences, Warrington, PA; stock solution 1 mg/ml). 48 h post- transfection whole cell extract was prepared for Western blot analysis, MMR assay or confocal laser microscopy. Plasmids Used pcDNA3.1+/MLH1, pcDNA3.1+/PMS2, pECFP-C1/ MLH1 (MLH1-GFP-N) and pDSRed-C1/PMS2 (PMS2-Red-N) PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31863