Screening of glycosylase activity on oxidative derivatives of methylcytosine: Pedobacter heparinus SMUG2 as a formylcytosine- and carboxylcytosine-DNA glycosylase

Abstract

5-Methylcytosine (mC) is an epigenetic mark that impacts transcription, development, diseases including cancer and aging. The demethylation process involves Tet-mediated stepwise oxidation of mC to hmC, fC, or caC, excision of fC or caC by thymine-DNA glycosylase (TDG), and subsequent base excision repair. Thymine-DNA glycosylase (TDG) belongs to uracil-DNA glycosylase (UDG) superfamily, which is a group of enzymes that are initially found to be responsible for excising the deaminated bases from DNA and generating apurinic/apyrimidinic (AP) sites. mC oxidative derivatives may also be generated from Fenton chemistry and γ-irradiation. In screening DNA glycosylase activity in UDG superfamily, we identified new activity on fC- and caC-containing DNA in family 2 MUG/TDG and family 6 HDG enzymes. Surprisingly, we found a glycosylase SMUG2 from bacterium Pedobacter heparinus (Phe), a subfamily of family 3 SMUG1 DNA glycosylase, displayed catalytic activity towards not only DNA containing uracil, but also fC and caC. Given the sequence and structural differences between the family 3 and other family enzymes, we investigated the catalytic mechanism using mutational, enzyme kinetics and molecular modeling approaches. Mutational analysis and kinetics measurements identified I62, N63 and F76 of motif 1, and H205 of motif 2 in Phe SMUG2 as important catalytic residues, of which H205 of motif 2 played a critical role in catalyzing the removal of fC and caC. A catalytic model underlying the roles of these residues was proposed. The structural and catalytic differences between Phe SMUG2 and human TDG were compared by molecular modeling and molecular dynamics simulations. This study expands our understanding of DNA glycosylase capacity in UDG superfamily and provides insights into the molecular mechanism of fC and caC excision in Phe SMUG2.