Fig. 2: Protein functional analysis.

A Schematic representation of the primary structure of PMS2 on the cDNA- and protein-level including the locations of the different missense variants investigated in this study. We tested one pathogenic control variant from the ATPase domain (p.Asp70Asn) and the four VUS of interest. intron-exon structure and annotations of functional motifs in the protein are given. B Schematic representation of the procedure for protein functional investigation. C Stability analysis by expression and western blotting. Expression plasmids for MLH1 and PMS2 (and its variants) were co-transfected in HEK293T cells. After 48 h, whole cell extracts were prepared and analyzed using SDS-PAGE and immunoblotting. β-Actin detection served as loading control. Expression levels of several experiments (3-8) in comparison to wildtype (%) were determined. Bars correspond to standard deviations. Controls and test variants are separated by a dashed line in the bar diagram. D Functional analysis by MMR (mismatch repair) assay. A test substrate with a G-T mismatch (indicated by triangles) within an EcoRV restriction site and a single-strand break (“nick”) directing repair to the open strand was incubated as detailed in Materials and Methods with nuclear extract of MLH1-PMS2-deficient HEK293T cells (50 µg) and complemented with extract containing the MLH1-PMS2 wildtype or variant as indicated (5 µg). After 15 min at 37 °C, the plasmid was isolated and analyzed by restriction digestion and agarose gel electrophoresis to test the functionality of the EcoRV restriction site. The appearance of two lower-weight fragments indicates repaired plasmid, while the upper band stems from unrepaired plasmid. Several independent assays were performed, and average repair values and standard deviations are shown in the bar diagram. Controls and test variants are separated by a dashed line in the bar diagram.