MUTYH cancer-associated variants within the interdomain connector differentially impact glycosylase activity and cellular DNA repair

The base excision repair (BER) glycosylase MUTYH initiates repair of 8-oxo-7,8-dihydroguanine (OG): adenine (A) mispairs to prevent G to T transversion mutations. Inherited biallelic mutations in <i>MUTYH</i> are correlated with the cancer pre-disposition syndrome <i>MUTYH</i> -associated polyposis (MAP) and contribute to an increased lifetime risk of colorectal cancer. Over 1,000 germline and somatic <i>MUTYH</i> variants have been reported that are associated with MAP and other cancers, but for most the functional impact is unknown. Herein, we examined a subset of cancer-associated variants (CAVs) localized in the interdomain connector (IDC), which links the N-terminal adenine excision and C-terminal OG recognition domains via its zinc linchpin motif and serves as a hub for downstream repair interactions. <i>In vitro</i> assays measuring glycosylase activity, lesion affinity, and AP endonuclease stimulation revealed no substantial defects relative to wild-type MUTYH. In contrast, a newly optimized mammalian cell assay revealed some IDC variants exhibit reduced repair. These results suggest that some variants disrupt steps downstream of adenine excision, whereas others impair lesion recognition and base excision. This work underscores the value of independent functional assays for accurately assessing variant dysfunction and classification. Analysis of <i>MUTYH</i> variants highlights the complexity of the roles of MUTYH in preserving genomic integrity.