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Alexander C Drohat - One of the best experts on this subject based on the ideXlab platform.
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excision of 5 carboxylcytosine by Thymine DNA Glycosylase
Journal of the American Chemical Society, 2019Co-Authors: L S Pidugu, Shuja Shafi Malik, Edwin Pozharski, Alexander C DrohatAbstract:5-Methylcytosine (mC) is an epigenetic mark that is written by methyltransferases, erased through passive and active mechanisms, and impacts transcription, development, diseases including cancer, and aging. Active DNA demethylation involves TET-mediated stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), or 5-carboxylcytosine (caC), excision of fC or caC by Thymine DNA Glycosylase (TDG), and subsequent base excision repair. Many elements of this essential process are poorly defined, including TDG excision of caC. To address this problem, we solved high-resolution structures of human TDG bound to DNA with cadC (5-carboxyl-2′-deoxycytidine) flipped into its active site. The structures unveil detailed enzyme–substrate interactions that mediate recognition and removal of caC, many involving water molecules. Importantly, two water molecules contact a carboxylate oxygen of caC and are poised to facilitate acid-catalyzed caC excision. Moreover, a substrate-dependent conformational change ...
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defining the impact of sumoylation on substrate binding and catalysis by Thymine DNA Glycosylase
Nucleic Acids Research, 2018Co-Authors: Christopher T Coey, Alexander C DrohatAbstract:: 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.
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structural basis of damage recognition by Thymine DNA Glycosylase key roles for n terminal residues
Nucleic Acids Research, 2016Co-Authors: Christopher T Coey, Alexander C Drohat, L S Pidugu, Shuja Shafi Malik, Edwin Pozharski, Kristen M VarneyAbstract:: 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 A) and TDG111-308 (1.71 A), revealing new enzyme-substrate contacts, direct and water-mediated. We also report a structure of the TDG82-308 product complex (1.70 A). 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.
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structural basis for excision of 5 formylcytosine by Thymine DNA Glycosylase
Biochemistry, 2016Co-Authors: L S Pidugu, Edwin Pozharski, Joshua W Flowers, Christopher T Coey, Marc M Greenberg, Alexander C DrohatAbstract: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.
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characterizing requirements for small ubiquitin like modifier sumo modification and binding on base excision repair activity of Thymine DNA Glycosylase in vivo
Journal of Biological Chemistry, 2016Co-Authors: Dylan Mclaughlin, Alexander C Drohat, Christopher T Coey, Weichih Yang, Michael J MatunisAbstract:: 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 G·caC 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.
Primo Schar - One of the best experts on this subject based on the ideXlab platform.
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oestrogen receptor β regulates epigenetic patterns at specific genomic loci through interaction with Thymine DNA Glycosylase
Epigenetics & Chromatin, 2016Co-Authors: Primo Schar, William Duong, Claudia Krawczyk, Nancy Bretschneider, Gabor Borbely, Mukesh Varshney, Christian Zinser, Joelle RueggAbstract:Background DNA methylation is one way to encode epigenetic information and plays a crucial role in regulating gene expression during embryonic development. DNA methylation marks are established by the DNA methyltransferases and, recently, a mechanism for active DNA demethylation has emerged involving the ten-eleven translocator proteins and Thymine DNA Glycosylase (TDG). However, so far it is not clear how these enzymes are recruited to, and regulate DNA methylation at, specific genomic loci. A number of studies imply that sequence-specific transcription factors are involved in targeting DNA methylation and demethylation processes. Oestrogen receptor beta (ERβ) is a ligand-inducible transcription factor regulating gene expression in response to the female sex hormone oestrogen. Previously, we found that ERβ deficiency results in changes in DNA methylation patterns at two gene promoters, implicating an involvement of ERβ in DNA methylation. In this study, we set out to explore this involvement on a genome-wide level, and to investigate the underlying mechanisms of this function.
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base excision by Thymine DNA Glycosylase mediates DNA directed cytotoxicity of 5 fluorouracil
PLOS Biology, 2009Co-Authors: Christophe Kunz, Teresa Lettieri, Yusuke Saito, Frauke Focke, David Schuermann, Jim Selfridge, Primo ScharAbstract:5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of Thymine DNA Glycosylase (TDG), one of four mammalian uracil DNA Glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU–induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU–based chemotherapy.
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the enigmatic Thymine DNA Glycosylase
DNA Repair, 2007Co-Authors: Daniel Cortazar, Christophe Kunz, Roland Steinacher, Yusuke Saito, Primo ScharAbstract:When it was first isolated from extracts of HeLa cells in Josef Jiricny's laboratory, the Thymine DNA Glycosylase (TDG) attracted attention because of its ability to remove Thymine, i.e. a normal DNA base, from G.T mispairs. This implicated a function of DNA base excision repair in the restoration of G.C base pairs following the deamination of a 5-methylcytosine. TDG turned out to be the founding member of a newly emerging family of mismatch-directed uracil-DNA Glycosylases, the MUG proteins, that act on a comparably broad spectrum of base lesion including G.U as the common, most efficiently processed substrate. However, because of its apparent catalytic inefficiency, some have considered TDG a poor DNA repair enzyme without an important biological function. Others have reported 5-meC DNA Glycosylase activity to be associated with TDG, thrusting the enzyme into limelight as a possible DNA demethylase. Yet others have found the Glycosylase to interact with transcription factors, implicating a function in gene regulation, which appears to be critically important in developmental processes. This article reviews all these developments in view of possible biological functions of this multifaceted DNA Glycosylase.
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functionality of human Thymine DNA Glycosylase requires sumo regulated changes in protein conformation
Current Biology, 2005Co-Authors: Roland Steinacher, Primo ScharAbstract:BACKGROUND: Base excision repair initiated by human Thymine-DNA Glycosylase (TDG) results in the generation of abasic sites (AP sites) in DNA. TDG remains bound to this unstable repair intermediate, indicating that its transmission to the downstream-acting AP endonuclease is a coordinated process. Previously, we established that posttranslational modification of TDG with Small Ubiquitin-like MOdifiers (SUMOs) facilitates the dissociation of the DNA Glycosylase from the product AP site, but the underlying molecular mechanism remained unclear. RESULTS: We now show that upon DNA interaction, TDG undergoes a dramatic conformational change, which involves its flexible N-terminal domain and accounts for the nonspecific DNA binding ability of the enzyme. This function is required for efficient processing of the G.T mismatch but then cooperates with the specific DNA contacts established in the active site pocket of TDG to prevent its dissociation from the product AP site after base release. SUMO1 conjugation to the C-terminal K330 of TDG modulates the DNA binding function of the N terminus to induce dissociation of the Glycosylase from the AP site while it leaves the catalytic properties of base release in the active site pocket of the enzyme unaffected. CONCLUSION: Our data provide insight into the molecular mechanism of SUMO modification mediated modulation of enzymatic properties of TDG. A conformational change, involving the N-terminal domain of TDG, provides unspecific DNA interactions that facilitate processing of a wider spectrum of substrates at the expense of enzymatic turnover. SUMOylation then reverses this structural change in the product bound TDG.
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t g mismatch specific Thymine DNA Glycosylase potentiates transcription of estrogen regulated genes through direct interaction with estrogen receptor α
Journal of Biological Chemistry, 2003Co-Authors: Dongsheng Chen, Pierre Chambon, Regine Losson, Marie J Lucey, Fladia Phoenix, Jorge Lopezgarcia, Stephen M Hart, L Buluwela, Charles R Coombes, Primo ScharAbstract:Nuclear receptors (NR) classically regulate gene expression by stimulating transcription upon binding to their cognate ligands. It is now well established that NR-mediated transcriptional activation requires the recruitment of coregulator complexes, which facilitate recruitment of the basal transcription machinery through direct interactions with the basal transcription machinery and/or through chromatin remodeling. However, a number of recently described NR coactivators have been implicated in cross-talk with other nuclear processes including RNA splicing and DNA repair. T:G mismatch-specific Thymine DNA Glycosylase (TDG) is required for base excision repair of deaminated methylcytosine. Here we show that TDG is a coactivator for estrogen receptor (ER). We demonstrate that TDG interacts with ER in vitro and in vivo and suggest a separate role for TDG to its established role in DNA repair. We show that this involves helix 12 of ER. The region of interaction in TDG is mapped to a putative -helical motif containing a motif distinct from but similar to the LXXLL motif that mediates interaction with NR. Together with recent reports linking TFIIH in regulating NR function, our findings provide new data to further support an important link between DNA repair proteins and nuclear receptor function.
Josef Jiricny - One of the best experts on this subject based on the ideXlab platform.
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DNA cytosine demethylation are we getting close
Cell, 2008Co-Authors: Josef Jiricny, Mirco MenigattiAbstract:Whether 5-methylcytosine (meC) can be enzymatically removed from vertebrate DNA has been the subject of extensive study and also some controversy. Rai et al. (2008) now report that cytosine demethylation can be accomplished in a one-cell zebrafish embryo by the combined action of a cytidine deaminase and a Thymine DNA Glycosylase.
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the versatile Thymine DNA Glycosylase a comparative characterization of the human drosophila and fission yeast orthologs
Nucleic Acids Research, 2003Co-Authors: Ulrike Hardeland, Josef Jiricny, Marl Bentele, Primo ScharAbstract:Human Thymine-DNA Glycosylase (TDG) is well known to excise Thymine and uracil from G.T and G.U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA Glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species-specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4- ethenocytosine opposite guanine and 5-fluorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and Thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5'-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA Glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual species.
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modification of the human Thymine DNA Glycosylase by ubiquitin like proteins facilitates enzymatic turnover
The EMBO Journal, 2002Co-Authors: Ulrike Hardeland, Roland Steinacher, Josef Jiricny, Primo ScharAbstract:DNA Glycosylases initiate base excision repair (BER) through the generation of potentially harmful abasic sites (AP sites) in DNA. Human Thymine-DNA Glycosylase (TDG) is a mismatch-specific uracil/Thymine-DNA Glycosylase with an implicated function in the restoration of G·C base pairs at sites of cytosine or 5-methylcytosine deamination. The rate-limiting step in the action of TDG in vitro is its dissociation from the product AP site, suggesting the existence of a specific enzyme release mechanism in vivo. We show here that TDG interacts with and is covalently modified by the ubiquitin-like proteins SUMO-1 and SUMO-2/3. SUMO conjugation dramatically reduces the DNA substrate and AP site binding affinity of TDG, and this is associated with a significant increase in enzymatic turnover in reactions with a G·U substrate and the loss of G·T processing activity. Sumoylation also potentiates the stimulatory effect of APE1 on TDG. These observations implicate a function of sumoylation in the controlled dissociation of TDG from the AP site and open up novel perspectives for the understanding of the molecular mechanisms coordinating the early steps of BER.
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Thymine DNA Glycosylase
Progress in Nucleic Acid Research and Molecular Biology, 2001Co-Authors: Ulrike Hardeland, Roland Steinacher, Teresa Lettieri, Josef Jiricny, Marl Bentele, Primo ScharAbstract:More than 50% of colon cancer-associated mutations in the p53 tumor suppressor gene are C--
Thymine DNA Glycosylase (TDG). Human TDG was discovered as an enzyme that has the potential to specifically remove Thymine and uracil bases mispaired with guanine through hydrolysis of their N-glycosidic bond, thereby generating abasic sites in DNA and initiating a base excision repair reaction. The same protein was later found to interact physically and functionally with the retinoid receptors RAR and RXR, and this implicated an unexpected function of TDG in nuclear receptor-mediated transcriptional activation of gene expression. The objective of this chapter is to put together the results of different lines of experimentation that have explored the Thymine DNA Glycosylase since its discovery and to critically evaluate their implications for possible physiological roles of this enzyme. -
Thymine DNA Glycosylase.
Progress in Nucleic Acid Research and Molecular Biology, 2001Co-Authors: Ulrike Hardeland, Roland Steinacher, Teresa Lettieri, Josef Jiricny, Marl Bentele, Primo ScharAbstract:More than 50% of colon cancer-associated mutations in the p53 tumor suppressor gene are C--
Atanu Maiti - One of the best experts on this subject based on the ideXlab platform.
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lesion search and recognition by Thymine DNA Glycosylase revealed by single molecule imaging
Nucleic Acids Research, 2015Co-Authors: Claudia N Buechner, Alexander C Drohat, Atanu Maiti, Ingrid TessmerAbstract:The ability of DNA Glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excisionrepair(BER).Here,weusesinglemoleculeimaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human Thymine DNA Glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG‐DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second Glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of Glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA Glycosylases.
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e2 mediated small ubiquitin like modifier sumo modification of Thymine DNA Glycosylase is efficient but not selective for the enzyme product complex
Journal of Biological Chemistry, 2014Co-Authors: Christopher T Coey, Atanu Maiti, Megan E Fitzgerald, Michael J Matunis, Katherine H Reiter, Catherine M Guzzo, Alexander C DrohatAbstract:Abstract Thymine DNA Glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (kobs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of kobs on TDG concentration gives kmax = 1.6 min−1 and K1/2 = 0.55 μm, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (Kd ∼50 nm). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.
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Thymine DNA Glycosylase can rapidly excise 5 formylcytosine and 5 carboxylcytosine potential implications for active demethylation of cpg sites
Journal of Biological Chemistry, 2011Co-Authors: Atanu Maiti, Alexander C DrohatAbstract:Thymine DNA Glycosylase (TDG) excises T from G·T mispairs and is thought to initiate base excision repair (BER) of deaminated 5-methylcytosine (mC). Recent studies show that TDG, including its Glycosylase activity, is essential for active DNA demethylation and embryonic development. These and other findings suggest that active demethylation could involve mC deamination by a deaminase, giving a G·T mispair followed by TDG-initiated BER. An alternative proposal is that demethylation could involve iterative oxidation of mC to 5-hydroxymethylcytosine (hmC) and then to 5-formylcytosine (fC) and 5-carboxylcytosine (caC), mediated by a Tet (ten eleven translocation) enzyme, with conversion of caC to C by a putative decarboxylase. Our previous studies suggest that TDG could excise fC and caC from DNA, which could provide another potential demethylation mechanism. We show here that TDG rapidly removes fC, with higher activity than for G·T mispairs, and has substantial caC excision activity, yet it cannot remove hmC. TDG excision of fC and caC, oxidation products of mC, is consistent with its strong specificity for excising bases from a CpG context. Our findings reveal a remarkable new aspect of specificity for TDG, inform its catalytic mechanism, and suggest that TDG could protect against fC-induced mutagenesis. The results also suggest a new potential mechanism for active DNA demethylation, involving TDG excision of Tet-produced fC (or caC) and subsequent BER. Such a mechanism obviates the need for a decarboxylase and is consistent with findings that TDG Glycosylase activity is essential for active demethylation and embryonic development, as are mechanisms involving TDG excision of deaminated mC or hmC.
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dependence of substrate binding and catalysis on ph ionic strength and temperature for Thymine DNA Glycosylase insights into recognition and processing of g t mispairs
DNA Repair, 2011Co-Authors: Atanu Maiti, Alexander C DrohatAbstract:Abstract Repair of G·T mismatches arising from deamination of 5-methylcytosine (m5C) involves excision of Thymine and restoration of a G·C pair via base excision repair (BER). Thymine DNA Glycosylase (TDG) is one of two mammalian enzymes that can specifically remove Thymine from G·T mispairs. While TDG can excise other bases, it maintains stringent specificity for a CpG context, suggesting deaminated m5C is an important biological substrate. Recent studies reveal TDG is essential for embryogenesis; it helps to maintain an active chromatin complex and initiates BER to counter aberrant de novo CpG methylation, which may involve excision of actively deaminated m5C. The relatively weak G·T activity of TDG has been implicated in the hypermutability of CpG sites, which largely involves C → T transitions arising from m5C deamination. Thus, it is important to understand how TDG recognizes and process substrates, particularly G·T mispairs. Here, we extend our detailed studies of TDG by examining the dependence of substrate binding and catalysis on pH, ionic strength, and temperature. Catalytic activity is relatively constant for pH 5.5–9, but falls sharply for pH > 9 due to severely weakened substrate binding, and, potentially, ionization of the target base. Substrate binding and catalysis diminish sharply with increasing ionic strength, particularly for G·T substrates, due partly to effects on nucleotide flipping. TDG aggregates rapidly and irreversibly at 37 °C, but can be stabilized by specific and nonspecific DNA. The temperature dependence of catalysis reveals large and unexpected differences for G·U and G·T substrates, where G·T activity exhibits much steeper temperature dependence. The results suggest that reversible nucleotide flipping is much more rapid for G·T substrates, consistent with our previous findings that steric effects limit the active-site lifetime of Thymine, which may account for the relatively weak G·T activity. Our findings provide important insight into catalysis by TDG, particularly for mutagenic G·T mispairs.
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stoichiometry and affinity for Thymine DNA Glycosylase binding to specific and nonspecific DNA
Nucleic Acids Research, 2011Co-Authors: Michael T Morgan, Atanu Maiti, Megan E Fitzgerald, Alexander C DrohatAbstract:Deamination of 5-methylcytosine to Thymine creates mutagenic G·T mispairs, contributing to cancer and genetic disease. Thymine DNA Glycosylase (TDG) removes Thymine from these G·T lesions, and follow-on base excision repair yields a G·C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G·U activity, but its role in G·T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G·U mispairs and abasic (G·AP) sites, and somewhat less tightly G·T mispairs. Kinetics experiments show 1:1 binding provides full G·T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G·T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G·U or G·T lesions.
Christopher T Coey - One of the best experts on this subject based on the ideXlab platform.
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defining the impact of sumoylation on substrate binding and catalysis by Thymine DNA Glycosylase
Nucleic Acids Research, 2018Co-Authors: Christopher T Coey, Alexander C DrohatAbstract:: 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.
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structural basis of damage recognition by Thymine DNA Glycosylase key roles for n terminal residues
Nucleic Acids Research, 2016Co-Authors: Christopher T Coey, Alexander C Drohat, L S Pidugu, Shuja Shafi Malik, Edwin Pozharski, Kristen M VarneyAbstract:: 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 A) and TDG111-308 (1.71 A), revealing new enzyme-substrate contacts, direct and water-mediated. We also report a structure of the TDG82-308 product complex (1.70 A). 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.
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structural basis for excision of 5 formylcytosine by Thymine DNA Glycosylase
Biochemistry, 2016Co-Authors: L S Pidugu, Edwin Pozharski, Joshua W Flowers, Christopher T Coey, Marc M Greenberg, Alexander C DrohatAbstract: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.
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characterizing requirements for small ubiquitin like modifier sumo modification and binding on base excision repair activity of Thymine DNA Glycosylase in vivo
Journal of Biological Chemistry, 2016Co-Authors: Dylan Mclaughlin, Alexander C Drohat, Christopher T Coey, Weichih Yang, Michael J MatunisAbstract:: 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 G·caC 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.
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Thymine DNA Glycosylase exhibits negligible affinity for nucleobases that it removes from DNA
Nucleic Acids Research, 2015Co-Authors: Shuja Shafi Malik, Alexander C Drohat, Edwin Pozharski, Christopher T Coey, Kristen M VarneyAbstract:: 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 A) 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.