MUTYH-mediated repair of oxidative DNA damage: Functional characterization and investigation into post-translational modifications of MUTYH and variants implicated in colon cancer

MUTYH Associated Polyposis (MAP) is a colorectal cancer predisposition syndrome that is caused by inherited biallelic mutations in the Base Excision Repair (BER) gene, MUTYH. MUTYH is a DNA glycosylase that removes adenine (A) misinserted opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG). In order to gain insights into MUTYH-mediated repair of OG:A mismatches and the disease mechanism of MAP, investigation into the functional activity of six MAP variants (Y165C, R227W, V232F, Q324R, G382D and P391L MUTYH) was carried out in the present study. WT MUTYH and MAP variants were overexpressed in E. coli and kinetically characterized using a correction for active fraction to control for differences due to overexpression and purification of the enzymes. All variants demonstrated a 25-40% reduction in the intrinsic glycosylase activity compared with WT MUTYH. When compared with WT MUTYH, all variants except for Q324R MUTYH showed a lower active enzyme yield after purification, a reduced ability to initiate MUTYH-mediated repair in a plasmid-based cellular repair assay, and the inability to complement for the absence of functional E. coli MutY in rifampicin resistance assays. Taken together, the in vitro glycosylase assays and cell-based assays reveal that the active enzyme yield and intrinsic glycosylase activity of MAP variants are important factors that might affect the MUTYH-dependent BER process.;To gain further insights into the biological activity of MUTYH, a baculovirus-driven insect cell expression system (BEVS) was designed and developed to overexpress MUTYH. WT MUTYH and the two most common MAP variants, Y165C and G382D MUTYH were purified at significantly higher levels in BEVS compared with the bacterial overexpression system. Both variants showed a binding defect while Y165C MUTYH also displayed a deficient glycosylase activity compared with WT MUTYH. WT MUTYH was analyzed for potential posttranslational modifications using mass spectrometry. An in vivo phosphorylation site was validated at Serine 524, which is located in a sequence known to be involved in interaction with Proliferating Cell Nuclear Antigen (PCNA). Characterization of the phosphomimetic (S524D) and phosphoablating (S524A) mutants suggests that phosphorylation at Ser 524 might affect MUTYH activity by altering the active fraction and stability of the enzyme.