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Thymine DNA glycosylase (TDG) is an essential enzyme involved in numerous biological pathways, including DNA repair, DNA demethylation, and transcriptional activation. Despite these important functions, the mechanisms surrounding the actions and regulation of TDG are poorly understood. In this study, we demonstrate that TDG induces phase separation of DNA and nucleosome arrays under physiologically relevant conditions in vitro and show that the resulting chromatin droplets exhibited behaviors typical of phase-separated liquids, supporting a liquid-liquid phase separation model. We also provide evidence that TDG has the capacity to form phase-separated condensates in the cell nucleus. The ability of TDG to induce chromatin phase separation is dependent on its intrinsically disordered N- and C-terminal domains, which in isolation, promote the formation of chromatin-containing droplets having distinct physical properties, consistent with their unique mechanistic roles in the phase separation process. Interestingly, DNA methylation alters the phase behavior of the disordered domains of TDG and compromises formation of chromatin condensates by full-length TDG, indicating that DNA methylation regulates the assembly and coalescence of TDG-mediated condensates. Overall, our results shed new light on the formation and physical nature of TDG-mediated chromatin condensates, which have broad implications for the mechanism and regulation of TDG and its associated genomic processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Thymine DNA glycosylase (TDG) is a multifaceted enzyme involved in several critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair. Recent studies have established regulatory relationships between TDG and RNA, but the molecular interactions underlying these relationships are poorly understood. Herein, we now demonstrate that TDG binds directly to RNA with nanomolar affinity. Using synthetic oligonucleotides of defined length and sequence, we show that TDG has a strong preference for binding G-rich sequences in single-stranded RNA but binds weakly to single-stranded DNA and duplex RNA. TDG also binds tightly to endogenous RNA sequences. Studies with truncated proteins indicate that TDG binds RNA primarily through its structured catalytic domain and that its disordered C-terminal domain plays a key role in regulating TDG's affinity and selectivity for RNA. Finally, we show that RNA competes with DNA for binding to TDG, resulting in the inhibition of TDG-mediated excision in the presence of RNA. Together, this work provides support for and insights into a mechanism wherein TDG-mediated processes (e.g., DNA demethylation) are regulated through the direct interactions of TDG with RNA., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Oxidation of DNA bases generates mutagenic and cytotoxic lesions that are implicated in cancer and other diseases. Oxidative base lesions, including 7,8-dihydro-8-oxoguanine, are typically removed through base excision repair. In addition, oxidized deoxynucleotides such as 8-oxo-dGTP are depleted by sanitizing enzymes to preclude DNA incorporation. While pathways that counter threats posed by 7,8-dihydro-8-oxoguanine are well characterized, mechanisms protecting against the major adenine oxidation product, 7,8-dihydro-8-oxoadenine (oxoA), are poorly understood. Human DNA polymerases incorporate dGTP or dCTP opposite oxoA, producing mispairs that can cause A→C or A→G mutations. oxoA also perturbs the activity of enzymes acting on DNA and causes interstrand crosslinks. To inform mechanisms for oxoA repair, we characterized oxoA excision by human thymine DNA glycosylase (TDG), an enzyme known to remove modified pyrimidines, including deaminated and oxidized forms of cytosine and 5-methylcystosine. Strikingly, TDG excises oxoA from G⋅oxoA, A⋅oxoA, or C⋅oxoA pairs much more rapidly than it acts on the established pyrimidine substrates, whereas it exhibits comparable activity for T⋅oxoA and pyrimidine substrates. The oxoA activity depends strongly on base pairing and is 370-fold higher for G⋅oxoA versus T⋅oxoA pairs. The intrinsically disordered regions of TDG contribute minimally to oxoA excision, whereas two conserved residues (N140 and N191) are catalytically essential. Escherichia coli mismatch-specific uracil DNA-glycosylase lacks significant oxoA activity, exhibiting excision rates 4 to 5 orders of magnitude below that of its ortholog, TDG. Our results reveal oxoA as an unexpectedly efficient purine substrate for TDG and underscore the large evolutionary divergence of TDG and mismatch-specific uracil DNA-glycosylase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts., Competing Interests: Conflict of interest A provisional patent application has been filed for hyTDG by the University of Texas. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Eukaryotic DNA methylation prevents genomic instability by regulating the expression of oncogenes and tumor-suppressor genes. The negative effects of dysregulated DNA methylation are highlighted by a strong correlation between mutations in the de novo DNA methyltransferase gene DNA methyltransferase 3 α ( DNMT3A ) and poor prognoses among acute myeloid leukemia (AML) patients. We show here that clinically observed DNMT3A mutations dramatically alter enzymatic activity, including mutations that lead to 6-fold hypermethylation and 3-fold hypomethylation of the human cyclin-dependent kinase inhibitor 2B ( CDKN2B or p15 ) gene promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN-AML) constitutes 40-50% of all AML cases and is the most epigenetically diverse AML subtype with pronounced changes in non-CpG DNA methylation. We identified a subset of DNMT3A mutations that enhance the enzyme's ability to perform non-CpG methylation by 2-8-fold. Many of these mutations mapped to DNMT3A regions known to interact with proteins that themselves contribute to AML, such as thymine DNA glycosylase (TDG). Using functional mapping of TDG-DNMT3A interactions, we provide evidence that TDG and DNMT3-like (DNMT3L) bind distinct regions of DNMT3A. Furthermore, DNMT3A mutations caused diverse changes in the ability of TDG and DNMT3L to affect DNMT3A function. Cell-based studies of one of these DNMT3A mutations (S714C) replicated the enzymatic studies and revealed that it causes dramatic losses of genome-wide methylation. In summary, mutations in DNMT3A lead to diverse levels of activity, interactions with epigenetic machinery components and cellular changes., (© 2019 Sandoval et al.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The E3 ubiquitin ligase CRL4(Cdt2) targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4(Cdt2) while binding to PCNA. In vertebrates, CRL4(Cdt2) promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4(Cdt2) targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4(Cdt2). Our results expand the group of vertebrate CRL4(Cdt2) substrates to include a bona fide DNA repair enzyme., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Thymine DNA glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation, and DNA demethylation. TDG mediates the cytotoxicity of the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the proteasomal pathway. Here we show that CRL4(Cdt2) E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S phase in a reaction that is dependent on the interaction of TDG with proliferating cell nuclear antigen (PCNA). siRNA-mediated depletion of PCNA or components of CRL4(Cdt2), specifically cullin4A/B or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA or change critical basic residues essential for the action of the PIP degron prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP degron is required for targeting TDG to the CRL4(Cdt2) E3 ubiquitin ligase complex. Compared with forced expression of wild type TDG, CRL4(Cdt2)- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus, CRL4(Cdt2)-dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA, and this degradation is important for preventing toxicity from excess TDG., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Uracil is an unavoidable aberrant base in DNA, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires a succession of intermediate products, including an abasic site and a single strand break, before the original DNA structure can be reconstituted. These repair intermediates are harmful for DNA replication and also interfere with transcription under cell-free conditions. However, their relevance for cellular transcription has not been proved. Here we investigated the influence of uracil incorporated into a reporter vector on gene expression in human cells. The expression constructs contained a single uracil opposite an adenine (to mimic dUTP misincorporation during DNA synthesis) or a guanine (imitating a product of spontaneous cytosine deamination). We found no evidence for a direct transcription arrest by uracil in either of the two settings because the vectors containing the base modification exhibited unaltered levels of enhanced GFP reporter gene expression at early times after delivery to cells. However, the gene expression showed a progressive decline during subsequent hours. In the case of U:A pairs, this effect was retarded significantly by knockdown of UNG1/2 but not by knockdown of SMUG1 or thymine-DNA glycosylase uracil-DNA glycosylases, proving that it is base excision by UNG1/2 that perturbs transcription of the affected gene. By contrast, the decline of expression of the U:G constructs was not influenced by either UNG1/2, SMUG1, or thymine-DNA glycosylase knockdown, strongly suggesting that there are substantial mechanistic or kinetic differences between the processing of U:A and U:G lesions in cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Wnt signaling plays an important role in colorectal cancer (CRC). Although the mechanisms of β-catenin degradation have been well studied, the mechanism by which β-catenin activates transcription is still not fully understood. While screening a panel of DNA demethylases, we found that thymine DNA glycosylase (TDG) up-regulated Wnt signaling. TDG interacts with the transcription factor TCF4 and coactivator CREB-binding protein/p300 in the Wnt pathway. Knocking down TDG by shRNAs inhibited the proliferation of CRC cells in vitro and in vivo. In CRC patients, TDG levels were significantly higher in tumor tissues than in the adjacent normal tissues. These results suggest that TDG warrants consideration as a potential biomarker for CRC and as a target for CRC treatment.

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.

Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G.T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G.C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2'-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn(140), implicated in the chemical step, and Arg(275), implicated in nucleotide flipping. The N140A variant binds substrate DNA with the same tight affinity as wild-type TDG, but it has no detectable base excision activity for a G.T substrate, and its excision rate is vastly diminished (by approximately 10(4.4)-fold) for G.U, G.FU, and G.BrU substrates. Thus, Asn(140) does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by approximately 6 kcal/mol (compared with 11.6 kcal/mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg(275) penetrates the DNA minor groove, filling the void created by nucleotide flipping. We found that the R275A and R275L substitutions weaken substrate binding and substantially decrease the base excision rate for G.T and G.BrU substrates. Our results indicate that Arg(275) promotes and/or stabilizes nucleotide flipping, a role that is most important for target nucleotides that are relatively large (dT and bromodeoxyuridine) and/or have a stable N-glycosylic bond (dT). Arg(275) does not contribute substantially to the binding of TDG to abasic DNA product, and it cannot account for the slow product release exhibited by TDG.

Myocardin is a serum response factor (SRF) co-activator that regulates transcription of many smooth muscle-specific genes and is essential for development of vascular smooth muscle. We used a yeast two-hybrid screen, with myocardin as bait in a search for factors that regulate myocardin transcriptional activity. From this screen, thymine DNA glycosylase (TDG) was identified as a myocardin-associated protein. TDG was originally identified as an enzyme involved in base excision repair of T:G mismatches caused by spontaneous deamination of methylated cytosines. However, TDG has also been shown to act as a transcriptional co-activator or co-repressor. The interaction between TDG and myocardin was confirmed in vitro by glutathione S-transferase pull down and in vivo by co-immunoprecipitation assays. We found that TDG abrogates myocardin induced expression of smooth muscle-specific genes and represses the trans-activation of the promoters of myocardin of these genes. Overexpression of TDG in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous TDG in SMCs increased smooth muscle-specific myosin heavy chain (SM MHC) and Telokin gene expression. Glutathione S-transferase pull-down assays demonstrated that TDG binds to a region of myocardin that includes the SRF binding domain. Furthermore, TDG was found to compete with SRF for binding to myocardin in vitro and in vivo, suggesting that TDG can inhibit expression of smooth muscle-specific genes, at least in part, through disrupting SRF/myocardin interactions. Finally, we demonstrated that the glycosylase activity of TDG is not required for its inhibitory effects on myocardin function. This study reveals a previously unsuspected role for the repair enzyme TDG as a repressor of smooth muscle differentiation via competing with SRF for binding to myocardin.

DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing apurinic/apyrimidinic (AP) DNA. For many glycosylases, the AP-DNA remains tightly bound, impeding enzymatic turnover. A prominent example is thymine DNA glycosylase (TDG), which removes T from G.T mispairs and recognizes other lesions, with specificity for damage at CpG dinucleotides. TDG turnover is very slow; its activity appears to reach a plateau as the [product]/[enzyme] ratio approaches unity. The follow-on base excision repair enzyme, AP endonuclease 1 (APE1), stimulates the turnover of TDG and other glycosylases, involving a mechanism that remains largely unknown. We examined the catalytic activity of human TDG (hTDG), alone and with human APE1 (hAPE1), using pre-steady-state kinetics and a coupled-enzyme (hTDG-hAPE1) fluorescence assay. hTDG turnover is exceedingly slow for G.T (k(cat)=0.00034 min(-1)) and G.U (k(cat)=0.005 min(-1)) substrates, much slower than k(max) from single turnover experiments, confirming that AP-DNA release is rate-limiting. We find unexpectedly large differences in k(cat) for G.T, G.U, and G.FU substrates, indicating the excised base remains trapped in the product complex by AP-DNA. hAPE1 increases hTDG turnover by 42- and 26-fold for G.T and G.U substrates, the first quantitative measure of the effect of hAPE1. hAPE1 stimulates hTDG by disrupting the product complex rather than merely depleting (endonucleolytically) the AP-DNA. The enhancement is greater for hTDG catalytic core (residues 111-308 of 410), indicating the N- and C-terminal domains are dispensable for stimulatory interactions with hAPE1. Potential mechanisms for hAPE1 disruption of the of hTDG product complex are discussed.

The repair of the multitude of single-base lesions formed daily in cells of all living organisms is accomplished primarily by the base excision repair pathway that initiates repair through a series of lesion-selective glycosylases. In this article, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the Escherichia coli mispaired uracil glycosylase (MUG). The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation and with the size and electronic inductive properties of a series of uracil 5-substituents. Data are presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, whereas the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over 5 orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases; however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases that focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome.

Thymine DNA glycosylase (TDG) excises thymine from G.T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG.X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG.FU, CpG.ClU, and CpG.BrU than for CpG.T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (k(max)) for G.X substrates depends significantly on the 5' base pair. For example, k(max) decreases by 6-, 11-, and 82-fold for TpG.ClU, GpG.ClU, and ApG.ClU, respectively, as compared with CpG.ClU. For the other G.X substrates, the 5'-neighbor effects have a similar trend but vary in magnitude. The activity for G.FU, G.ClU, and G.BrU, with any 5'-flanking pair, meets and in most cases significantly exceeds the CpG.T activity. Strikingly, human TDG activity is reduced 10(2.3)-10(4.3)-fold for A.X relative to G.X pairs and reduced further for A.X pairs with a 5' pair other than C.G. The effect of altering the 5' pair and/or the opposing base (G.X versus A.X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed.

Deinococcus radiodurans is extremely resistant to the effects of ionizing radiation. The source of the radiation resistance is not known, but an expansion of specific protein families related to stress response and damage control has been observed. DNA repair enzymes are among the expanded protein families in D. radiodurans, and genes encoding five different uracil-DNA glycosylases are identified in the genome. Here we report the three-dimensional structure of the mismatch-specific uracil-DNA glycosylase (MUG) from D. radiodurans (drMUG) to a resolution of 1.75 angstroms. Structural analyses suggest that drMUG possesses a novel catalytic residue, Asp-93. Activity measurements show that drMUG has a modified and broadened substrate specificity compared with Escherichia coli MUG. The importance of Asp-93 for activity was confirmed by structural analysis and abolished activity for the mutant drMUGD93A. Two other microorganisms, Bradyrhizobium japonicum and Rhodopseudomonas palustris, possess genes that encode MUGs with the highest sequence identity to drMUG among all of the bacterial MUGs examined. A phylogenetic analysis indicates that these three MUGs form a new MUG/thymidine-DNA glycosylase subfamily, here called the MUG2 family. We suggest that the novel catalytic residue (Asp-93) has evolved to provide drMUG with broad substrate specificity to increase the DNA repair repertoire of D. radiodurans.

SUMO-1 is a member of a family of ubiquitin-like molecules that are post-translationally conjugated to various cellular proteins in a process that is mechanistically similar to ubiquitylation. To identify molecules that bind noncovalently to SUMO-1, we performed yeast two-hybrid screening with a SUMO-1 mutant that cannot be conjugated to target proteins as the bait. This screening resulted in the isolation of cDNAs encoding the b isoform of thymine DNA glycosylase (TDGb). A deletion mutant of TDGb (TDGb(Delta11)) that lacks a region shown to be required for noncovalent binding of SUMO-1 was also found not to be susceptible to SUMO-1 conjugation at an adjacent lysine residue, suggesting that such binding is required for covalent modification. In contrast, another mutant of TDGb (TDGb(KR)) in which the lysine residue targeted for SUMO-1 conjugation is replaced with arginine retained the ability to bind SUMO-1 non-covalently. TDGb was shown to interact with the promyelocytic leukemia protein (PML) in vitro as well as to colocalize with this protein to nuclear bodies in transfected cells. TDGb(KR) also colocalized with PML, whereas TDGb(Delta11) did not, indicating that the noncovalent SUMO-1 binding activity of TDGb is required for colocalization with PML. Furthermore, SUMO-1 modification of TDGb and PML enhanced the interaction between the two proteins. These results suggest that SUMO-1 functions to tether proteins to PML-containing nuclear bodies through post-translational modification and noncovalent protein-protein interaction.