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Thymine DNA Glycosylase (TDG): The excised base is not retained in the enzyme‐product complex
Author(s) -
Malik Shuja,
Pozharisky Edwin,
Varney Kristen,
Drohat Alexander
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.710.13
Subject(s) - dna glycosylase , base excision repair , dna , dna demethylation , dna repair , ternary complex , biochemistry , deamination , chemistry , biology , enzyme , gene , dna methylation , gene expression
Thymine DNA Glycosylase (TDG) is an enzyme that initiates base excision repair (BER) pathway and has important functions in maintaining genetic integrity and modulating epigenetic regulation. TDG was discovered as a repair enzyme that removes T from G:T mismatches that arise by deamination of 5‐methylcytosine (mC). TDG also has a critical role in active DNA demethylation; it excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), epigenetic bases that are generated via oxidation of 5mC by Tet (ten eleven translocation) enzymes. TDG also excises other lesions, including uracil (U) and 5‐hydroxymethyluracil (hmU). Like most DNA glycosylases, TDG exhibits very tight binding and slow dissociation from its reaction product, abasic DNA. In contrast, TDG has negligible affinity for, and is not inhibited by, the nucleobases that it excises from DNA. Intriguingly, it was reported that TDG retains the hmU base that it excises from a G:hmU substrate in a ternary product complex (TDG‐DNA‐base). While a ternary product complex was observed in crystal structures of some glycosylases (UNG, TAG), a binary (Enzyme‐DNA) complex was observed for MUG, the most‐closely related to glycosylase to TDG. We solved high‐resolution crystal structures (up to 1.6 Å) of TDG product complexes generated from several different substrates (G:hmU, G:U, G:T, G:fC, G:caC) and find no evidence that the excised base is retained in the active site. Moreover, NMR chemical shift perturbation ( 15 N‐HSQC) experiments collected for multiple TDG product complexes give the same result. Taken together, our findings indicate that TDG has little affinity for the bases that it removes from DNA. Implications for catalysis by TDG and other glycosylases will be discussed.