The enigmatic thymine DNA glycosylase (original) (raw)
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Cloning and Expression of Human G/T Mismatch-specific Thymine-DNA Glycosylase
Journal of Biological Chemistry, 1996
Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of baseexcision repair enzymes.
Nucleic Acids Research, 2003
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 signi®cant facts. (i) All eukaryotic TDG orthologs have broad and species-speci®c substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most ef®ciently processed substrates of all are uracil and 3,N4ethenocytosine opposite guanine and 5-¯uorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and thymine derivatives are processed with an appreciable ef®ciency 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 ®ndings advance our current view on the role of TDG proteins and document that they have evolved with high structural exibility to counter a broad range of DNA base damage in accordance with the speci®c needs of individual species.
Journal of Biological Chemistry
Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of baseexcision repair enzymes.
Cell, 2011
DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally-and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5hydroxymethyluracil excision repair.
2003
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 spon-taneous deamination of 5-methylcytosine and cyto-sine. 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 signi®cant facts. (i) All eukaryotic TDG orthologs have broad and species-speci®c substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most ef®ciently processed substrates of all are uracil and 3,N4-ethenocytosine opposite guanine and 5-¯uorouracil in any double-stranded DNA context; (i...
Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase
Uracil-DNA glycosylase (UDG) protects the genome by removing mutagenic uracil residues resulting from deamination of cytosine. Uracil binds in a rigid pocket at the base of the DNA-binding groove of human UDG and the specificity for uracil over the structurally related DNA bases thymine and cytosine is conferred by shape complementarity, as well as by main chain and Asn2O4 side chain hydrogen bonds. Here we show that replacement of Asn2O4 by Asp or Tyrl47 by Ala, Cys or Ser results in enzymes that have cytosine-DNA glycosylase (CDG) activity or thymine-DNA glycosylase (TDG) activity, respectively. CDG and the TDG all retain some UDG activity. CDG and TDG have kcat values in the same range as typical multisubstrate-DNA glycosylases, that is at least three orders of magnitude lower than that of the highly selective and efficient wild-type UDG. Expression of CDG or TDG in Escherichia coli causes 4to 100-fold increases in the yield of rifampicin-resistant mutants. Thus, single amino acid substitutions in UDG result in less selective DNA glycosylases that release normal pyrimidines and confer a mutator phenotype upon the cell. Three of the four new pyrimidine-DNA glycosylases resulted from single nucleotide substitutions, events that may also happen in vivo. Keywords: cytosine-DNA glycosylase/human uracil-DNA glycosylase/mutator enzymes/site-directed mutagenesis/thymine-DNA glycosylase et al., 1995) UDGs were recently solved. These studies demonstrated that the structure of the active site groove of UDG is very conserved, in agreement with the high degree of sequence conservation between UDGs from different prokaryotic and eukaryotic sources (Olsen et al., 1989; Mol et al., 1995a). Uracil binds in a rigid pocket at the base of the DNA-binding groove and the absolute specificity for uracil over the structurally related bases thymine and cytosine is conferred by shape complementarity, as well as by main chain and Asn2O4 side chain hydrogen bonds (Mol et al., 1995a; Savva et al., 1995). For human UDG, four amino acid residues (Asn2O4, Gln144, Asp145 and His268) were found to be critical for UDG activity (Mol et al., 1995a). These are all located in the uracil-binding pocket. His268 is critical for catalysis, but not for uracil recognition. Asn2O4 is critical for substrate binding and forms hydrogen bonds with 04 and N3 of uracil through side chain amide-N and-0, respectively. Tyrl47 also lines the active site uracilbinding pocket and contributes to shape complementarity. The position of its side chain excludes binding of pyrimidines carrying substitutions in the C5-position, such as methyl. It is well established that inactivating mutations in genes for enzymes involved in nucleotide-excision repair as well as mismatch repair, are associated with increased risk of mutations and cancer development (reviewed in Friedberg et al., 1995). DNA glycosylases initiate repair by the third major DNA repair pathway; the base-excision repair pathway. DNA glycosylases are widely expressed in human tissues and a significant interindividual variation in expression has been observed (Mymes et al., 1983). However, no link between low expression of such enzymes and human disease has been established. In this paper we demonstrate that certain mutations in the active site of human UDG result in novel enzymatic activities that release normal pyrimidines from DNA and cause mutations.
Nucleic Acids Research, 2014
The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N 6 -ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes.
The Thymine−DNA Glycosylase Regulatory Domain: Residual Structure and DNA Binding †
Biochemistry, 2008
Thymine-DNA glycosylases (TDGs) initiate base excision repair by debasification of the erroneous thymine or uracil nucleotide in G · T and G · U mispairs which arise at high frequency through spontaneous or enzymatic deamination of methylcytosine and cytosine, respectively. Human TDG has furthermore been shown to have a functional role in transcription and epigenetic regulation through the interaction with transcription factors from the nuclear receptor superfamily, transcriptional coregulators, and a DNA methyltransferase. The TDG N-terminus encodes regulatory functions, as it assures both G · T versus G · U specificity and contains the sites for interaction and posttranslational modification by transcription-related activities. While the molecular function of the evolutionarily conserved central catalytic domain of TDG in base excision repair has been elucidated by determination of its three-dimensional structure, the mechanisms by which the N-terminus exerts its regulatory roles, as well as the function of TDG in transcription regulation, remain to be understood. We describe here the residual structure of the TDG N-terminus in both contexts of the isolated domain and the entire protein. These studies lead to the characterization of a small structural domain in the TDG N-terminal region preceding the catalytic core and coinciding with the region of functional regulation of TDG's activities. This regulatory domain exhibits a small degree of organization and is implicated in dynamic molecular interactions with the catalytic domain and nonselective interactions with double-stranded DNA, providing a molecular explanation for the evolutionarily acquired G · T mismatch processing activity of TDG.
The Role of Thymine DNA Glycosylase in Transcription, Active DNA Demethylation, and Cancer
Cancers, 2022
DNA methylation is an essential covalent modification that is required for growth and development. Once considered to be a relatively stable epigenetic mark, many studies have established that DNA methylation is dynamic. The 5-methylcytosine (5-mC) mark can be removed through active DNA demethylation in which 5-mC is converted to an unmodified cytosine through an oxidative pathway coupled to base excision repair (BER). The BER enzyme Thymine DNA Glycosylase (TDG) plays a key role in active DNA demethylation by excising intermediates of 5-mC generated by this process. TDG acts as a key player in transcriptional regulation through its interactions with various nuclear receptors and transcription factors, in addition to its involvement in classical BER and active DNA demethylation, which serve to protect the stability of the genome and epigenome, respectively. Recent animal studies have identified a connection between the loss of Tdg and the onset of tumorigenesis. In this review, we s...