Fig 2: Functional assay for MUTYH variants. A: Germline mutations observed in MUTYH related to MAP. Alignment of E. coli MutY and human MUTYH is shown; 47 mutations observed in MUTYH related to MAP are indicated. B: Suppression of spontaneous mutations by expression of wild-type MUTYH and 47 variants in E. coli CC104mutY. The control shows transfectants with empty vectors. Green line shows the cut-off value between functionally retained and partially defective. Red line shows the cut-off value between functionally defective and partially defective.

Fig 3: Simultaneous suppression of MTH1 and MYH was efficiently achieved using a two-vector system. (a) Steps of cell line establishment are illustrated here. Top 15% of cells expressing GFP and RFP670 were sorted to improve the knockdown efficiency. Expression levels of MYH and MTH1 were analyzed after 96 h of treatment with Dox in shRNA set1 (b) and shRNA set2 (c) using qRT–PCR analysis. Data were normalized to NT-shRNA expressing cells and presented as mean±s.e.m. from three independent experiments in triplicate. To investigate the knockdown efficiency at protein level, western blot analysis was performed after 96 h of Dox treatment of cells with shRNA set1 (d) and shRNA set2 (e). Data were double normalized with GAPDH and NT-shRNA control samples and expressed as percentage. The graphs represent mean±s.e.m. from three independent experiments. P-values were calculated using one-way analysis of variance (ANOVA). *P<0.05, **P<0.01, ***P<0.001, ****P⩽0.0001, NS, not significant.

Fig 4: Mapping of MUTYH variants on protein structure. A: Structure of B. stearothermophilus MutY bound to DNA. The iron and sulfur molecules in the [4Fe-4S] cluster are shown as yellow and gray spheres, respectively. B: Structure of MUTYH simulated by homology modeling. C: Mapping of amino acid residues examined in this study and functional information. Red sites show the residues with defective substitution. Yellow sites show the residues with partially defective substitution. Green sites show the residues with retained substitutions. p.R245, p.P405, p.R437, and p.A473 are also highlighted in yellow, as the residues with multiple substitutions that are defective and retained.

Fig 5: Western blot analyses of 10 representative MUTYH variants. A FLAG sequence was added at the N-terminus of MUTYH gene. Expressed protein in HCT116 cells detected by anti-Flag antibody. The ratios of the variants to the wild type are shown as the relative MUTYH protein level.