Impaired 8-Hydroxyguanine Repair Activity of MUTYH Variant p.Arg109Trp Found in a Japanese Patient with Early-Onset Colorectal Cancer
Figure 2
Comparison of the DNA glycosylase activity of WT MUTYH and p.Arg81Trp variant MUTYH protein using a DNA cleavage assay. (a) Purification of WT, p.Arg81Trp, and p.Asp208Asn MUTYH type 2 recombinant proteins. The MUTYH proteins were overexpressed and purified using the pET system and TALON metal affinity resins. Representative results for the expression and purification of MUTYH proteins resolved by SDS-PAGE and stained with CBB are shown. “−” and “+” mean the absence and presence, respectively, of IPTG induction, and “Pur” means purified MUTYH type 2 proteins. The arrow points to the MUTYH-His6 protein band. (b) Western blot of purified MUTYH type 2 proteins. MUTYH-His6 proteins are indicated by the arrow. Purified recombinant DNA glycosylase NEIL1-His6 protein, which was previously prepared using the same system as that used for the MUTYH-His6 protein [38], was included as a negative control. (c) Substrate used in the DNA cleavage assay. 32P-labeled 30-mer double-stranded oligonucleotides containing or not containing a single 8OHG mispair were prepared. (d) Measurement of the DNA glycosylase activity of WT, p.Arg81Trp, and p.Asp208Asn MUTYH type 2 protein on double-stranded DNA containing an 8OHG using the DNA cleavage assay. The reaction mixture was subjected to 20% PAGE. The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. “M” means a marker oligonucleotide. The amount of cleavage products as a proportion of the total oligonucleotides was calculated as the % incision, and the values are shown in (e). The values are the means ± standard errors of data from three independent experiments. ND means not detected.
