C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins

Abstract

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.

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 ±S.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.

Figure 2. Influence of fluorescent labeling of MutLα on protein expression levels.

To analyze the effect of fluorescent dyes on protein expression levels, HEK293T cells were transiently cotransfected with different MutLα constructs (see below) and (A) Western blot analysis was carried out after 48 h using anti-MLH1 or anti-PMS2, respectively, controlled by β-actin detection. (B) Amounts of expressed proteins were assessed by measuring the signal intensities of protein bands with Multi Gauge V3.2 software. Graphs indicate the results (mean ±S.D.) of at least four independent experiments in which the proportion of protein expression using an unbiased method were presented. 1: negative control (untransfected). 2: MLH1/PMS2 unlabeled; 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.

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.

Figure 4. Influence of fluorescent tags on MMR activity of MutLα.

HEK293T cells were transiently cotransfected with various labeled or unlabeld MutLα constructs and 48 h post transfection MMR activity of different MutLαs were assessed in vitro in parallel with unlabeled MutLα by quantifying the 3′-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 . 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 MutLα was set at 100 percent and repair of fluorescent tagged MutLα was determined in relation to the wild-type sample that was expressed, processed and tested in parallel. Average repair values and standard deviations (±) were determined from four independent experiments. Single PMS2 tagged MutLαs, single MLH1-GFP-N tagged MutLαs 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.

Figure 5. Putative three-dimensional structure models of fluorescent labeled MutLα.

Using PyMol (Warren DeLano, http://www.pymol.org/), GFP or Red fluorescent proteins were attached to (A) N-termini of MLH1 and PMS2 or (B) C-termini of MutLα. C-terminal tags seem to hide the C-terminal region of MutLα and consequently might avoid DNA interaction. (C) Corresponding amino acid sequences of linker regions are shown. The dashed line between PMS2 and the N-terminal fluorescent tag illustrates a putative α-helix (unknown structure) of the first thirty amino acids of PMS2.