Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G·T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5-formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes. Here, we report structural and functional studies of TDG82-308, a new construct containing 29 more N-terminal residues than TDG111-308, the construct used for previous structures of DNA-bound TDG. Crystal structures and NMR experiments demonstrate that most of these N-terminal residues are disordered, for substrate- or product-bound TDG82-308. Nevertheless, G·T substrate affinity and glycosylase activity of TDG82-308 greatly exceeds that of TDG111-308 and is equivalent to full-length TDG. We report the first high-resolution structures of TDG in an enzyme-substrate complex, for G·U bound to TDG82-308 (1.54 Å) and TDG111-308 (1.71 Å), revealing new enzyme-substrate contacts, direct and water-mediated. We also report a structure of the TDG82-308 product complex (1.70 Å). TDG82-308 forms unique enzyme–DNA interactions, supporting its value for structure-function studies. The results advance understanding of how TDG recognizes and removes modified bases from DNA, particularly those resulting from deamination.

Thymine DNA glycosylase (TDG) is a base excision repair enzyme with key functions in epige-netic 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 solved a crystal structure of TDG bound to DNA with a non-cleavable (2′-fluoroarabino) analog 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.

Base excision repair (BER) is one of several DNA repair pathways found in all three domains of life. BER counters the mutagenic and cytotoxic effects of damage that occurs continuously to the nitrogenous bases in DNA, and its critical role in maintaining genomic integrity is well established. However, BER also performs essential functions in processes other than DNA repair, where it acts on naturally modified bases in DNA. A prominent example is the central role of BER in mediating active DNA demethylation, a multi-step process that erases the epigenetic mark, 5-methylcytosine (5mC), or derivatives thereof, converting them back to cytosine. Here, we review recent advances in the understanding of how BER mediates this critical component of epigenetic regulation, in plants and animals.

Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G·T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten–eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme–product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme–substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme–product complex.

Thymine-DNA glycosylase (TDG) plays critical roles in DNA base excision repair and DNA demethylation. It has been proposed, based on structural studies and in vitro biochemistry, that sumoylation is required for efficient TDG enzymatic turnover following base excision. However, whether sumoylation is required for TDG activity in vivo has not previously been tested. We have developed an in vivo assay for TDG activity that takes advantage of its recently discovered role in DNA demethylation and selective recognition and repair of 5-carboxylcytosine. Using this assay, we investigated the role of sumoylation in regulating TDG activity through the use of TDG mutants defective for sumoylation and Small Ubiquitin-like Modifier (SUMO) binding and by altering TDG sumoylation through SUMO and SUMO protease overexpression experiments. Our findings indicate that sumoylation and SUMO binding are not essential for TDG-mediated excision and repair of 5-carboxylcytosine bases. Moreover, in vitro assays revealed that apurinic/apyrimidinic nuclease 1 provides nearly maximum stimulation of TDG processing of G⅐caC substrates. Thus, under our assay conditions, apurinic/apyrimidinic nuclease 1-mediated stimulation or other mechanisms sufficiently alleviate TDG product inhibition and promote its enzymatic turnover in vivo.Regulation and coordination of DNA repair mechanisms is essential for maintaining genome integrity and proper cell function. Sumoylation is a post-translational protein modification critical for DNA damage repair, including repair of DNA single-and double-stranded breaks, interstrand cross-links, and nucleotide mismatches (1, 2). Although affecting repair processes through a variety of distinct mechanisms, primary functions of sumoylation at DNA lesions include promotion of protein-protein interactions and stimulation of protein extraction or turnover (1, 3). The exact molecular effects of sumoylation on many modified repair factors, however, remain to be fully understood.Thymine-DNA glycosylase (TDG) 3 is a monofunctional glycosylase involved in DNA repair, DNA demethylation, and transcription activation (4 -6). The best studied role of TDG is in base excision repair (BER), where it specifically recognizes G/U and G/T mismatches arising from spontaneous deamination of cytosine or 5-methylcytosine, respectively. BER proceeds through a process involving glycosylase recognition of a specific DNA lesion and removal of the lesion to produce an apurinic/apyrimidinic (AP) site. TDG-mediated BER is of particular interest because of the observed product inhibition caused by high affinity binding of TDG to the AP site following base excision (7,8). This product inhibition can be relieved by apurinic/apyrimidinic endonuclease 1 (APE1), the enzyme that acts immediately downstream of TDG in the BER pathway (8, 9). Alternatively, in vitro studies and structural analyses have suggested an intriguing model for overcoming product inhibition involving TDG sumoylation (10 -14).TDG is sumoylated at a single lysine residue near its C te...

Background: Post-translational SUMO modification of TDG weakens its DNA binding and was proposed to regulate dissociation of a tight enzyme-product complex. Results: In vitro sumoylation of TDG by SUMO-1 and SUMO-2 is efficient for free and DNA-bound TDG. Conclusion: E2-mediated sumoylation is not selective for product-bound TDG but could potentially stimulate product release. Significance: Our findings inform the mechanism and role of TDG sumoylation.

Base excision repair (BER) is a conserved and ubiquitous pathway that is initiated by DNA glycosylases, which recognize and remove damaged or mismatched nucleobases, setting the stage for restoration of the correct DNA sequence by follow-on BER enzymes. DNA glycosylases employ a nucleotide-flipping step prior to cleavage of the N-glycosyl bond, and most exhibit slow release of the abasic DNA product and/or strong product inhibition. As such, studying the catalytic mechanism of these enzymes requires care in the design, execution, and interpretation of single- and multiple-turnover kinetics experiments, which is the topic of this chapter.

Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC) and it removes two mC derivatives, 5−formylcytosine (fC) and 5−carboxylcytosine (caC), in a multistep pathway for DNA demethylation. TDG is modified by small ubiquitin-like modifier (SUMO) proteins, but the impact of sumoylation on TDG activity is poorly defined and the functions of TDG sumoylation remain unclear. We determined the effect of TDG sumoylation, by SUMO-1 or SUMO-2, on substrate binding and catalytic parameters. Single turnover experiments reveal that sumoylation dramatically impairs TDG base-excision activity, such that G·T activity is reduced by ≥45-fold and fC and caC are excised slowly, with a reaction half-life of ≥9 min (37°C). Fluorescence anisotropy studies reveal that unmodified TDG binds tightly to G·fC and G·caC substrates, with dissociation constants in the low nanomolar range. While sumoylation of TDG weakens substrate binding, the residual affinity is substantial and is comparable to that of biochemically-characterized readers of fC and caC. Our findings raise the possibility that sumoylation enables TDG to function, at least transiently, as reader of fC and caC. Notably, sumoylation could potentially facilitate TDG recruitment of other proteins, including transcription factors or epigenetic regulators, to these sites in DNA.