The versatile thymine DNA-glycosylase: a comparative characterization of the human, Drosophila and ®ssion yeast orthologs (original) (raw)
Related papers
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.
The enigmatic thymine DNA glycosylase
Dna Repair, 2007
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.
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.
Proceedings of the National Academy of Sciences, 1998
Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3,N 4 -ethenocytosine (C), 1,N 6 -ethenoadenine (A), N 2 ,3-ethenoguanine, and 1,N 2 -ethenoguanine cause mutations. The A residues are excised by the human and the Escherichia coli 3-methyladenine-DNA glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repairing C residues have not yet been described. We have identified two homologous proteins present in human cells and E. coli that remove C residues by a DNA glycosylase activity. The human enzyme is an activity of the mismatch-specific thymine-DNA glycosylase (hTDG). The bacterial enzyme is the double-stranded uracil-DNA glycosylase (dsUDG) that is the homologue of the hTDG. In addition to uracil and C-DNA glycosylase activity, the dsUDG protein repairs thymine in a G͞T mismatch. The fact that C is recognized and efficiently excised by the E. coli dsUDG and hTDG proteins in vitro suggests that these enzymes may be responsible for the repair of this mutagenic lesion in vivo and be important contributors to genetic stability.
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.
Specificity of Human Thymine DNA Glycosylase Depends on N -Glycosidic Bond Stability
Journal of The American Chemical Society, 2006
Initiating the DNA base excision repair pathway, DNA glycosylases find and hydrolytically excise damaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multiple forms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G·T mispairs and recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the millionfold excess of normal G·C pairs over G·T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here, we examine the possibility that hTDG specificity depends on the stability of the scissile base-sugar bond by determining the maximal activity (k max ) against a series of nucleobases with varying leaving group ability. We find that hTDG removes 5-fluorouracil 78-fold faster than uracil and 5-chlorouracil 572-fold faster than thymine, differences that can be attributed predominantly to leaving group ability. Moreover, hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5hydroxycytosine, indicating that cytosine has access to the active site. A plot of log(k max ) versus leaving group pK a reveals a Brønsted-type linear free energy relationship with a large negative slope of β lg = −1.6 ± 0.2, consistent with a highly dissociative reaction mechanism. Further, we find that the hydrophobic active site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity. Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site.
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.
Enzymatic capture of an extrahelical thymine in the search for uracil in DNA
Nature, 2007
The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U*A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U*A or U*G base pairs in a background of approximately 10(9) T*A or C*G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T*A and U*A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.
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.