The versatile thymine DNA-glycosylase: a comparative characterization of the human, Drosophila and fission yeast orthologs

Abstract

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.

Figure 1

Conservation of MUG-type UDGs. (A) Shown are the phylogenetic relationships between representative UDGs of the α/β-fold superfamily. Included are Homo sapiens UNG2 (hsUNG2; accession no. P22674), TDG (hsTDG; Q13569) and SMUG1 (hsSMUG1; O95862); Drosophila melanogaster Thd1p (dmThd1p; Q9V4D8) and SMUG1 (dmSMUG1; Swiss-Prot, Q9VEM1); Xenopus laevis SMUG1 (xlSMUG1; Q9YGN6) Schizosaccharomyces pombe Ung1p (spUng1p; O74834) and Thp1p (spThp1p: O59825); Saccharomyces cerevisiae Ung1p (saccUng1p; P12887); Serratia marcescens Mug (smMug; P43343); Escherichia coli Ung (ecUng; P12295) and Mug (ecMug; P43342); Streptomyces coelicolor UDGb (scUDGb; NP_626251) and MUG (scMug; NP_625542); Pyrobaculum aerophilum UDGa (paUDGa; NP_558739) and UDGb (paUDGb: NP_559226); Thermus thermophilus UDG (tthUDG; CAD29337); Mycobacterium tuberculosis UDG (mtUDG; NP_335742); Thermotoga maritima UDG (tmUDG; NP_228321); and Archaeoglobus fulgidus UDG (afUDG; NP_071102). (B) Amino acid sequence alignment of the catalytic core domains of spThp1p, dmThd1p with hsTDG and ecMug. The conserved MUG characteristic active site motif G(I/L)NPG(L/I) in the N‐terminal part of the catalytic domain and the less conserved C-terminal residues that are critical for a specific interaction with the substrate DNA are framed in red; other conserved residues are framed and shaded. The arrow indicates the position of the critical catalytic residue (N140) of TDG (6). (C) Schematic representation of the overall domain organization of the same members of the MUG subfamily of UDGs. The conserved core domains of the proteins are shaded, and the relative positions of the active site motifs indicated. All sequence analyses were done with the ClustalW routine (Blosum30 matrix; Gap penalty, 10; Gap extension penalty, 0.1) of the MacVector Sequence analysis software (Version 7.1.1; Accelrys, Burlington, USA).

Figure 2

Glycosylase activity of TDG, Thd1p and Thp1p. (A) Fractions of purified TDG, Thd1p and Thp1p analyzed by SDS–PAGE. A 2 µg aliquot of each protein was loaded onto a 12.5% gel. The proteins were visualized by Coomassie brilliant blue staining. (B) The 60mer oligonucleotide duplex DNA used as substrate. An unlabeled upper strand oligonucleotide contains guanine or adenine at the position indicated. The complementary 5′-fluorescein-labeled (green asterisk) lower strand positions either a cytosine, a thymine, a uracil or any other target base of interest opposite the guanine as indicated. AccI and SalI digestions result in 22mer and 23mer product formation, respectively. (C) Thd1p and Thp1p generate APE1 and alkaline-sensitive AP-sites in a G·U substrate. Standard base release reactions were done in 20 µl volumes in the presence of 1 pmol of substrate DNA and 1 pmol of protein. The reaction products shown were analyzed on 15% denaturing polyacrylamide gels. The positions of the 60mer substrate DNA, the respective product fragments, and the 22mer and 23mer AccI and SalI restriction fragments are indicated.

Figure 3

Differential G·T processing by TDG, Thd1p and Thp1p. The ability to generate alkaline-sensitive sites in standard substrates was assayed for TDG (A), Thd1p (B) and Thp1p (C) in the absence or presence of 1 U of Ugi. Shown are the results obtained with 60mer dsDNA substrates containing either G·C, G·T, G·U or A·U base pairs or a single uracil in ssDNA at identical positions. All reactions were done in a 20 µl volume containing equimolar amounts (50 nM) of substrate DNA and enzyme, and the products were separated on 15% denaturing polyacrylamide gels. The positions of the 60mer substrate DNA and 23mer product fragment are indicated.

Figure 4

Kinetic properties of Thd1p and Thp1p in comparison with TDG. The time-dependent generation of alkaline-sensitive AP-sites was measured by incubation of TDG (A), Thd1p (B) and Thp1p (C) with double-stranded 60mer substrates containing either a single G·U, G·T or A·U mismatch, or ssDNA containing U as indicated. The substrate and enzyme concentrations were 50 nM as described for the standard TDG assay (6). All reactions were performed at 37°C and stopped after the indicated times by the addition of NaOH. Product formation was monitored and quantified after denaturing gel electrophoresis and fluorescent scanning. Shown are nicking efficiencies averaged from at least three independent experiments over short (upper panels) and longer (lower panels) time courses; standard deviations are listed in Table 1.

Figure 5

Substrate and product DNA-binding properties. Comparative EMSAs were performed with TDG, Thd1p and Thp1p as indicated. The DNA-binding reactions were performed in a 10 µl volume with 1 pmol of labeled homoduplex (G·C), substrate (G·T, G·U, A·U) or product DNA (G·AP, A·AP) in the presence of 10 pmol of unlabeled homoduplex competitor DNA and either 4 pmol of TDG, 4 pmol of Drosophila Thd1p or 5 pmol of S.pombe Thp1p. The AP-site-containing DNAs were generated by incubation of uracil substrate DNA with E.coli UDG and subsequent purification as described (6). Bound fluorescein-labeled DNA was separated from free substrate DNA in 6% native polyacrylamide gels. Shown are fluorograms of representative gels.

Figure 6

Substrate DNA bases examined. Shown are the chemical structures of purine and pyrimidine DNA bases with modifications relevant to this study.

Figure 7

Substrate processing ability of eukaryotic MUG proteins. The substrate processing efficiencies of TDG, Thd1p and Thp1p were determined in kinetic assays as described in the text. Each experiment was repeated at least three times, and the resulting P_max, T₅₀ and Eff_rel (= P_max/T₅₀) values are listed in Table 1. Graphically illustrated are the relative processing efficiencies (Eff_rel) obtained with dsDNA substrates showing comparable kinetic properties. For ease of comparison, the Eff_rel values are normalized to the relative efficiency of G·U processing for each individual enzyme [= Eff_rel(X·Y)/Eff_rel(G·U)].