The oxidized
guanine lesion
7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is highly mutagenic, resulting in G:C to T:A transversion mutations in the absence of repair. The Escherichia coli
adenine glycosylase MutY and its human homolog (hMYH) play an important role in the prevention of mutations associated with OG by removing misincorporated
adenine residues from OG:A mismatches. Previously, biallelic mutations of hMYH have been identified in a British family (Family N) with symptoms characteristic of
familial adenomatous polyposis (FAP), which is typically associated with mutations in the
adenomatous polyposis coli (APC) gene. Afflicted members of this family were compound heterozygotes for two mutations in hMYH, Y165C and G382D. These positions are highly conserved in MutY across phylogeny. The current work reveals a reduced ability of the hMYH variants compared to wild-type (WT) hMYH to
complement the activity of E.coli MutY in mutY((-)) E.coli. In vitro analysis of the corresponding mutations in E.coli MutY revealed a reduction in the
adenine glycosylase activity of the
enzymes. In addition, evaluation of substrate affinity using a substrate analog,
2'-deoxy-2'-fluoroadenosine (FA) revealed that both mutations severely diminish the ability to recognize FA, and discriminate between OG and G. Importantly,
adenine removal with both the mutant and WT E.coli
enzymes was observed to be less efficient from a mismatch in the sequence context observed to be predominantly mutated in
tumors of Family N. Interestingly, the magnitude of the reduced activity of the E.coli mutant
enzymes relative to the WT
enzyme was magnified in the "hotspot" sequence context. If the corresponding mutations in hMYH cause similar sensitivity to sequence context, this effect may contribute to the specific targeting of the APC gene. The lack of complementation of the hMYH variants for MutY, and the reduced activity of the Y82C and G253D E.coli
enzymes, provide additional circumstantial evidence that the somatic mutations in APC, and the occurrence of FAP in Family N, are due to a reduced ability of the Y165C and G382D hMYH
enzymes to recognize and repair OG:A mismatches.