Novel Activity of Escherichia coli Mismatch Uracil-DNA Glycosylase (Mug) Excising 8-(Hydroxymethyl)-3,N4-ethenocytosine, a Potential Product Resulting from Glycidaldehyde Reaction

Glycidaldehyde is an industrial chemical which has been shown to be genotoxic in in vitro experiments and carcinogenic in rodent studies. It is a bifunctional alkylating agent capable of reacting with DNA to form exocyclic hydroxymethyl-substituted ethenobases. In this work, 8-(hydroxymethyl)-3,N4-etheno-2'-deoxycytidine (8-HM-εdC), a potential nucleoside derivative of glycidaldehyde, was synthesized using phosphoramidite chemistry and site-specifically incorporated into a defined 25-mer oligodeoxynucleotide. The 8-HM-εC adduct is structurally related to 3,N4-ethenocytosine (εC), a product of reaction with vinyl chloride or through lipid peroxidation. In Escherichia coli, εC has been shown previously to be a primary substrate for the mismatch uracil-DNA glycosylase (Mug). In this study, we report that the same glycosylase also acts on 8-HM-εC in an oligonucleotide duplex. The enzyme binds to the 8-HM-εC-oligonucleotide to a similar extent as the εC-oligonucleotide. The Mug excision activity toward 8-HM-εC is ∼2.5-fold lower than that toward the εC substrate. Both activities can be stimulated up to ∼2-fold higher by the addition of E. coli endonuclease IV. These two adducts, when mispaired with normal bases, were all excised from DNA by Mug with similar efficiencies. Structural studies using molecular simulations showed similar adjustment and hydrogen bonding pattern for both 8-HM-εC·G and εC·G pairs in oligomer duplexes. We believe that these findings may have biological and structural implications in defining the role of 8-HM-εC in glycosylase recognition/repair.