Cancer-associated variants and a common polymorphism of MUTYH exhibit reduced repair of oxidative DNA damage using a GFP-based assay in mammalian cells

Biallelic germline mutations in the base excision repair enzyme gene MUTYH lead to multiple colorectal adenomas and carci-nomas referred to as MUTYH-associated polyposis. MUTYH removes adenine misincorporated opposite the DNA oxidation product, 8-oxoguanine (OG), thereby preventing accumula-tion of G:C to T:A transversion mutations. The most common cancer-associated MUTYH variant proteins when expressed in bacteria exhibit reduced OG:A mismatch affinity and adenine removal activity. However, direct evaluation of OG:A mismatch repair efficiency in mammalian cells has not been assessed due to the lack of an appropriate assay. To address this, we developed a novel fluorescence-based assay of OG:A repair and measured the repair capacity of MUTYH-associated polyposis variants expressed in Mutyh−/ − mouse embryonic fibroblasts (MEFs). The repair of a single site–specific synthetic lesion in a gree