Structural Basis for Excision of 5-Formylcytosine by Thymine DNA Glycosylase

Abstract

Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epigenetic regulation. Performing a critical step in a pathway for active DNA demethylation, TDG removes 5-formylcytosine and 5-carboxylcytosine, oxidized derivatives of 5-methylcytosine that are generated by TET (ten-eleven translocation) enzymes. We determined a crystal structure of TDG bound to DNA with a noncleavable (2'-fluoroarabino) analogue of 5-formyldeoxycytidine flipped into its active site, revealing how it recognizes and hydrolytically excises fC. Together with previous structural and biochemical findings, the results illustrate how TDG employs an adaptable active site to excise a broad variety of nucleobases from DNA.

Figure 1

DNA cytosine methylation by DNMT and DNA demethylation via the TET-TDG-BER pathway.

Figure 2

Structure of TDG^82-308 bound to G·fC DNA (PDB ID: 5T2W). TDG residues are white (N blue, O red), the dfC^F-containing DNA is yellow (2′-F is cyan), and water molecules are red spheres. A 2F_o-F_c electron density map, contoured at 1.0 σ, is shown for DNA (yellow) and water molecules (blue) but not the enzyme (for clarity). Dashed lines are hydrogen bonds, with interatomic distances (Å) shown for contacts to fC (all others are ≤3.5 Å).

Figure 3

Binding of the nucleophilic water molecule. TDG is in cartoon and stick format (white), the fdC nucleotide is yellow, and the nucleophile (water) is a red sphere (other waters omitted for clarity). A 2F_o-F_c map, contoured at 1.0 σ, is shown for the nucleophile. Dashed lines are H bonds, some with interatomic distances (Å). The distance from the nucleophile to C1′ of fdC^F (4.0 Å) is indicated.

Figure 4

Potential stepwise mechanism for TDG excision of fC from DNA. Interactions in the E ·S complex are those observed in the structure reported here. Rupture of the N-glycosyl bond yields a putative short-lived intermediate, with an fC anion and oxacarbenium ion; addition of the nucleophile (water) gives products (fC and an abasic site).

Figure 5

Superposition of structures to compare TDG interactions with (A) fC versus caC and (B) fC versus U (all are 2′-F-β analogs). (A) For the TDG^82-308-G·fC structure, DNA is yellow, protein is white, water molecules are red spheres, and hydrogen bonds are black dashes (distances ≤3.5 Å). For TDG^111-308-G·caC (PDB ID: 3UOB), DNA is cyan, protein is green, and hydrogen bonds are cyan dashes (no waters are observed). (B) For TDG^82-308-G·fC the coloring is the same as in panel A. For TDG^82-308-G·U (PDB ID: 5HF7), DNA is cyan, protein is green, water molecules are light cyan, and hydrogen bonds are cyan. In both panels, the putative nucleophilic water molecule(s) is marked (*).

Scheme 1. Synthesis of the phosphoramidite for 2′-fluoroarabino-5-formyldeoxycytidine^a

^aKey: a) TBSCl b) Pd₂(dba)₃, PPh₃, Bu₃SnH, CO c) 1,3-Propanediol, triethyl orthoformate, TiCl₄ d) p-Methoxybenzoyl chloride e) HF•pyridine f) DMTCl g) Phosphitylation.

Chart 1

2′-F substitutions in 2′-deoxynucleotides