Human thymine DNA glycosylase

Bao Ngoc Dinh '25, Andrew Van Horn '25, and Ryan Yarcusko '25

Contents:

I. Introduction




Thymine DNA Glycosylase (TDG) serves as an epigenetic modification protein by erasing methylated cytosines. Among other purposes, researchers have found strong links between the lack of TDG in cells and certain cancers [1]. TDG of the DNA strand and orients the base for nucleophilic attack by H2O [2]. Once more, TDG serves to demethylate cytosines by removing derivatives of the methylated cytosines before DNA pol III adds an unmethylated cytosine in its place [1].


II. General Structure



is composed of 410 amino acid residues in total [2]. However, only the 227 residues shown here (residues 82-308) are necessary for glycosylase function. Nonetheless, there are alpha-helices and beta-sheets arranged asymmetrically.

Additionally, the catalytic domain is approximately 195 residues and centrally located. There are additional N and C-terminal disordered regions that are important for protein interactions and post-translational modifications, though very little of these non-catalytic regions are included in this PDB file .



III. Substrate


While TDG can act on thymine mismatches, its most common role in cells is to erase the methylation of cytosines [3]. However, TDG is unable to excise 5-methylcytosine . To convert mC to an apropriate, oxidized substrate, the ten-eleven translocation (TET) protein oxidizes mC to 5-hydroxymethylcytosine . The TET protein then further oxidizes hmC to 5-formylcytosine , or even further oxidizes fC to 5-carboxylcytosine . These oxidation steps are preparation steps for base excision because TDG only removes fC and caC cytosine derivatives.


Pathway

Figure 1. The transformations occuring along the pathway to 5-formylcytosine. Accronyms are Cytosine (C), 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), 5-carboxylcytosine (caC), DNA Methyl Transferase (DNMT), Ten-Eleven-Translocation Enzymes (TET), Base Excision Repair (BER). Pathway image from Pidugu et al. 2016 [3].


Additionally, there is a fluorine atom attached to the excised sugar of the nucleotide in our tutorial. This is because the added fluorine allows TDG to bind the excised base normally, but prevents TDG from actually excising the base. This was done in the study that the pdb file came from [2] so that they could crystallize how the base looks when it is "flipped out" into TDG:

IV. DNA Binding



TDG translocates across the DNA until reaching the lesion site [4]. At the lesion site, TDG penetrates deep within the minor groove and causes a dramatic curvature in the DNA (curvature not shown in this model). Once more, in reaching the lesion site, cationic residues of TDG form nonspecific interactions with anionic phosphate residues [2]. Further, there are many enzyme-substrate interactions that allow TDG to recognize and excise fC from DNA. For example, TDG provides a relatively short polar contact (2.8 Angstroms). This is the only polar contact provided to the fC formyl oxygen, and it is likely important for substrate binding and catalysis. likely accounts for the ability of TDG to bind tightly to DNA containing fC but not mC or hmC, or even T mismatches. As can be seen, the Tyr OH undergoes with the fC, as well as base-stacking interactions between Tyr-152 and fC. Additionally, the methyl of forms a nonpolar contact with the fC formyl carbon, helping

There are various water-mediated and other contacts involved in DNA -TDG interaction that aid fC binding and/or excision. The amino group of fC and a water molecule. Asn-191 is known to contribute to the binding fC in the enzyme - substrate complex. TDG provides 2 backbone N-H contacts to fC O2, one of which is fairly short (2.7 Angstroms). fC O2 is also contacted by .




V. Base-excision



Primarily, the oxidized forms of mC (caC, fC) can be excised by TDG, fC being excised most favorably. Experimental evidence shows that TDG catalyzes the N-glycosyl bond cleavage. This leads to a short-lived intermediate, which is attacked by a nucleophile (H2O) to create an abasic site [5]. TDG binds the putative in the enzyme - substrate complex: . To encourage the flipping of the excised base, the cationic side chain of TDG directly contacts each of the two anionic phosphates on the flanking nucleotides and forms an additional with the 5' phosphate of the excised base. Additionally, there are a variety of residues in the active site of TDG that stabilize the flipping of the excised base, but active site residues can vary slightly depending on the base in question.


can also be excised by TDG with similar activity to fC [6]. It is hypothesized that TDG could stabilize departing Uracil anion via hydrogen bond to O2 and O4 [2], which in turn suggests that the excised fC could also be stabilized by hydrogen bonds to O2 and the formyl oxygen [3].

Uracil Contacts

Figure 2. Thymine DNA glycosylase contacts on uracil [2].


This semi-specific active site allows the TDG to excise a variety of nucleotides. Important active site residues are organized into the table below [2,3].

Please click "Clear Protein and Lines" before using the table for optimal functionality




Amino Acids
 Direct Contacts to fC
 Water-mediated Contacts to fC
 Backbone Contacts


N/A
 N/A



N/A

N/A
 N/A

N/A
 N/A
N/A
 N/A

N/A
N/A


N/A

N/A
 N/A

N/A
N/A

N/A


N/A
 N/A

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VI. References

  1. Onabote, O., Hassan, H. M., Isovic, M., & Torchia, J. (2022). The Role of Thymine DNA Glycosylase in Transcription, Active DNA Demethylation, and Cancer. In Cancers (Vol. 14, Issue 3). MDPI. https://doi.org/10.3390/cancers14030765
  2. Coey, C. T., Malik, S. S., Pidugu, L. S., Varney, K. M., Pozharski, E., & Drohat, A. C. (2016). Structural basis of damage recognition by thymine DNA glycosylase: Key roles for N-terminal residues. Nucleic Acids Research, 44(21), 10248-10258. https://doi.org/10.1093/nar/gkw768
  3. Pidugu, L. S., Flowers, J. W., Coey, C. T., Pozharski, E., Greenberg, M. M., & Drohat, A. C. (2016). Structural Basis for Excision of 5-Formylcytosine by Thymine DNA Glycosylase. Biochemistry, 55(45), 6205-6208. https://doi.org/10.1021/acs.biochem.6b00982
  4. Tian J, Wang L, Da LT. Atomic resolution of short-range sliding dynamics of thymine DNA glycosylase along DNA minor-groove for lesion recognition. Nucleic Acids Res. 2021 Feb 22;49(3):1278-1293. doi: 10.1093/nar/gkaa1252. PMID: 33469643; PMCID: PMC7897493.
  5. Drohat, A. C., & Maiti, A. (2014). Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA. Organic and Biomolecular Chemistry, 12(42), 8367-8378. https://doi.org/10.1039/c4ob01063a
  6. Maiti, A., & Drohat, A. C. (2011). Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: Potential implications for active demethylation of CpG sites. Journal of Biological Chemistry, 286(41), 35334-35338. https://doi.org/10.1074/jbc.C111.284620











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