Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA

Abstract

Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.

Figure 1. Electrophoretic mobility shift assay of hTDG^cat(N140A) with 23mer dsDNA containing G•T, G•U, G•5fC and G•5caC pairs

(a) The proposed 5mC demethylation pathway. (b–c) The EMSA assay for hTDG^cat(N140A) with dsDNA containing G•5fC and G•5caC pairs. The experiments were performed with 4 nM ³²P-labeled DNA (40 pM ³²P-labeled DNA for G•AP pairs) and various concentrations of hTDG^cat(N140A) as shown.

Figure 2. Schematic diagram of the hTDG^cat(N140A)-A•5caC complex structure

(a) Overall structure of hTDG^cat(N140A) bound with 5caC-containing dsDNA. The 5caC and the wedge residue Arg275 are shown as sticks and colored in magenta and green, respectively. (b) A network of hydrogen bonds in the active site of hTDG specifically recognizes 5caC. Residues involved in the interactions are labeled and shown as sticks, and hydrogen bonds are shown as yellow dashes. (c) The interactions involved in the 5-carboxyl-binding pocket. The atoms involved are presented as transparent spheres. (d) Superposition of 5caC and β-F-5caC in the active site pocket of the hTDG^cat(N140A)-A•5caC and hTDG^cat-G• β-F-5caC structures. Residues involved in the interactions in the β-F-5caC structure are shown as sticks and colored in yellow and orange. The fluorine atom is labeled in dark green. The hydrogen bond interaction between β-F-5caC and residue Ser271 is shown in yellow dashes.