Crystal structure of a family 4 uracil-DNA glycosylase from Thermus thermophilus HB8

Uracil-DNA glycosylase (UDG; EC 3.2.2.-) removes uracil from DNA to initiate DNA base excision repair. Since hydrolytic deamination of cytosine to uracil is one of the most frequent DNA-damaging events in all cells, UDG is an essential enzyme for maintaining the integrity of genomic information. For the first time, we report the crystal structure of a family 4 UDG from Thermus thermophilus HB8 (TthUDG) complexed with uracil, solved at 1.5 angstroms resolution. As opposed to UDG enzymes in its other families, TthUDG possesses a [4Fe-4S] cluster. This iron-sulfur cluster, which is distant from the active site, interacts with loop structures and has been suggested to be unessential to the activity but necessary for stabilizing the loop structures. In addition to the iron-sulfur cluster, salt-bridges and ion pairs on the molecular surface and the presence of proline on loops and turns is thought to contribute to the enzyme's thermostability. Despite very low levels of sequence identity with Escherichia coli and human UDGs (family 1) and E.coli G:T/U mismatch-specific DNA glycosylase (MUG) (family 2), the topology and order of secondary structures of TthUDG are similar to those of these distant relatives. Furthermore, the coordinates of the core structure formed by beta-strands are almost the same. Positive charge is distributed over the active-site groove, where TthUDG would bind DNA strands, as do UDG enzymes in other families. TthUDG recognizes uracil specifically in the same manner as does human UDG (family 1), rather than guanine in the complementary strand DNA, as does E.coli MUG (family 2). These results suggest that the mechanism by which family 4 UDGs remove uracils from DNA is similar to that of family 1 enzymes.     

Correlated cleavage of single- and double-stranded substrates by uracil-DNA glycosylase

Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double- and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.     

Crystal structure of family 5 uracil-DNA glycosylase bound to DNA

Uracil-DNA glycosylase (UDG) removes uracil generated by the deamination of cytosine or misincorporation of deoxyuridine monophosphate. Within the UDG superfamily, a fifth UDG family lacks a polar residue in the active-site motif, which mediates the hydrolysis of the glycosidic bond by activation of a water molecule in UDG families 1-4. We have determined the crystal structure of a novel family 5 UDG from Thermus thermophilus HB8 complexed with DNA containing an abasic site. The active-site structure suggests this enzyme uses both steric force and water activation for its excision reaction. A conserved asparagine residue acts as a ligand to the catalytic water molecule. The structure also implies that another water molecule acts as a barrier during substrate recognition. Based on no significant open-closed conformational change upon binding to DNA, we propose a "slide-in" mechanism for initial damage recognition.     

Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo

The 3,N(4)-ethenocytosine (epsilon C) residue might have biological role in vivo since it is recognized and efficiently excised in vitro by the E. coli mismatch-specific uracil-DNA glycosylase (MUG) and the human thymine-DNA glycosylase (hTDG). In the present work we have generated mug defective mutant of E. coli by insertion of a kanamycin cassette to assess the role of MUG in vivo. We show that human TDG complements the enzymatic activity of MUG when expressed in a mug mutant. The epsilon C-DNA glycosylase defective strain did not exhibit spontaneous mutator phenotype and did not show unusual sensitivity to any of the following DNA damaging treatments: methylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet light, H(2)O(2), paraquat. However, plasmid DNA damaged by 2-chloroacetaldehyde treatment in vitro was inactivated at a greater rate in a mug mutant than in wild-type host, suggesting that MUG is required for the in vivo processing of the ethenobases. In addition, 2-chloroacetaldehyde treatment induces preferentially G.C --> C.G and A.T --> T.A transversions in mug mutant. Comparison of the mutation frequencies induced by the site-specifically incorporated epsilon C residue in E. coli wild-type versus mug indicates that MUG repairs more than 80% of epsilon C residues in vivo. Furthermore, the results show that nucleotide excision repair and recombination are not involved in the processing of epsilon C in E. coli. Based on the mutagenesis data we suggest that epsilon C may be less toxic and less mutagenic than expected. The increased spontaneous mutation rate for G.C --> A.T transition in the ung mug double mutant as compared to the single ung mutant suggest that MUG may be a back-up repair enzyme to the classic uracil-DNA glycosylase.     

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

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.     

Real-time monitoring of uracil removal by uracil-DNA glycosylase using fluorescent resonance energy transfer probes

As a highly conserved damage repair protein, uracil-DNA glycosylase (UDG) mainly catalyzes the excision of uracil from DNA to sustain the genome integrity. Here a novel method for monitoring the uracil removal in real time is introduced. Double-stranded DNA probes modified with uracil residues that can occur in fluorescent resonance energy transfer (FRET) were used as substrates and detecting probes in a homogeneous solution. This method not only overcame the drawbacks of traditional radioactive assays, such as discontinuity and being time-consuming and complicated, but also was used to accurately determine the kinetic constant of UDG. The limit of detection of UDG was 0.033 U/ml. The KM and Kcat were 0.11 microM and 4 s(-1), respectively. In addition, the method was applied to investigate the influence of chemical drugs on UDG activity. The results showed that 10 mM fluorouracil (5-FU) and gentamicin are inhibitors to UDG. The in vitro detection of UDG in A549 cells showed that the activity of UDG was four times greater after the cells were treated with cisplatin. These results showed that this method can monitor uracil removal in real time and conveniently assay UDG activity with ultrasensitivity and excellent specificity in the homogeneous solution. This method is also amenable to high-throughput drug screening in vitro.     

Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms

Abstract Hyperthermophiles exist in conditions which present an increased threat to the informational integrity of their DNA, particularly by hydrolytic damage. As in mesophilic organisms, specific activities must exist to restore and protect this template function of DNA. In this study we have demonstrated the presence of thermally stable uracil-DNA glycosylase activities in seven hyperthermophiles; one bacterial: Thermotoga maritima , and six archaeal: Sulfolobus solfataricus, Sulfolobus shibatae, Sulfolobus acidocaldarius, Thermococcus litoralis, Pyrococcus furiosus and Pyrobaculum islandicum . Uracil-DNA glycosylase inhibitor protein of the Bacillus subtilis bacteriophage PBS1 shows activity against all of these, suggesting a highly conserved tertiary structure between hyperthermophilic and mesophilic uracil-DNA glycosylases.     

Expression and purification of NEIL3, a human DNA glycosylase homolog

The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1–301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.     

An efficient cloning of DNA fragments by a method based on uracil-DNA glycosylase and endonuclease IV

We introduced a novel method to clone random DNA fragments independent of ligation reaction. The method involves the generation of long protruding ends on PCR amplification DNA. Both oligonucleotides used for the amplification of the vector DNA carried one uracil residue at the tenth position from the 5′ end and this made the creation of the 3′ protruding ends of linearized vector possible by uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). 76 groups of annealed oligonucleotides that had ten-nucleotides protruding at 3′-end, which were complementary to those at 3′-end of the linearized vector, were designed. The linearized vector and the annealed oligonucleotide were mixed together to transform E.coli directly without ligation reaction. The number of the clone that grew on the plates had been demonstrated to reach 1 × 10⁵ transformants/μg and 96.1% of transformants harbored the cloned fragments. From the results of transformation, we can confirm that the efficiency of the creation of 3′ protruding ends in our method is high and our cloning method is benefit to produce recombinants easily and efficiently.     

Base Excision Repair and its Role in Maintaining Genome Stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10⁴ events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Specificity of stimulation of human 8-oxoguanine-DNA glycosylase by AP endonuclease

Human 8-oxoguanine-DNA glycosylase OGG1 is an enzyme that removes abundant oxidative lesion 8-oxoguanine (8-oxoG) from DNA. Excision of 8-oxoG by OGG1 is inhibited by the abasic DNA reaction product and is stimulated by AP endonuclease APEX1. Besides 8-oxoG, OGG1 shows activity towards several other base lesions. Here we report that APEX1 efficiently stimulates OGG1 on good substrates (8-oxoadenine, 8-oxoinosine, or 6-methoxy-8-oxoguanine opposite to cytosine) but the stimulation is low or absent with poor OGG1 substrates (8-oxoadenine or 8-oxoinosine opposite to thymine; 8-oxoG or 8-aminoguanine opposite to adenine; 8-oxonebularine, 8-metoxyguanine, inosine or guanine opposite to cytosine). APEX1 significantly improves the ability of OGG1 to excise 8-aminoguanine from its naturally occurring pair with cytosine, making it possible that OGG1 repairs this lesion. Overall, APEX1 serves to improve specificity of OGG1 for its biologically relevant substrates.     

Repair of deaminated base damage by Schizosaccharomyces pombe thymine DNA glycosylase

Thymine DNA glycosylases (TDG) in eukaryotic organisms are known for their double-stranded glycosylase activity on guanine/uracil (G/U) base pairs. Schizosaccharomyces pombe (Spo) TDG is a member of the MUG/TDG family that belongs to a uracil DNA glycosylase superfamily. This work investigates the DNA repair activity of Spo TDG on all four deaminated bases: xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine, and uracil from cytosine. Unexpectedly, Spo TDG exhibits glycosylase activity on all deaminated bases in both double-stranded and single-stranded DNA in the descending order of X > I > U  O. In comparison, human TDG only excises deaminated bases from G/U and, to a much lower extent, A/U and G/I base pairs. Amino acid substitutions in motifs 1 and 2 of Spo TDG show a significant impact on deaminated base repair activity. The overall mutational effects are characterized by a loss of glycosylase activity on oxanine in all five mutants. L157I in motif 1 and G288M in motif 2 retain xanthine DNA glycosylase (XDG) activity but reduce excision of hypoxanthine and uracil, in particular in C/I, single-stranded hypoxanthine (ss-I), A/U, and single-stranded uracil (ss-U). A proline substitution at I289 in motif 2 causes a significant reduction in XDG activity and a loss of activity on C/I, ss-I, A/U, C/U, G/U, and ss-U. S291G only retains reduced activity on T/I and G/I base pairs. S163A can still excise hypoxanthine and uracil in mismatched base pairs but loses XDG activity, making it the closest mutant, functionally, to human TDG. The relationship among amino acid substitutions, binding affinity and base recognition is discussed.     

极端嗜热古菌尿嘧啶DNA糖苷酶的研究进展

高温会加快碱基脱氨基反应形成损伤碱基的速率,进一步对脱氨基的碱基进行复制会导致突变。因此,极端嗜热古菌基因组的稳定性面临着其生存高温环境的挑战。胞嘧啶脱氨基形成尿嘧啶,是常见的脱碱基类型,复制DNA中尿嘧啶会造成GC→AT的突变。尿嘧啶DNA糖苷酶(Uracil DNA glycosylase,UDG)是修复DNA中尿嘧啶的关键酶。基于识别底物的特异性,UDG分为6个家族,广泛分布在细菌、古菌、真核生物以及一些病毒中。基因组序列显示,极端嗜热古菌至少编码一种UDG。目前,对于细菌和真核生物的UDG已进行了大量的研究,但是关于极端嗜热古菌UDG的研究相对较少,尚处于初期阶段。本文综述了极端嗜热古菌UDG的研究进展,并对今后的研究提出了展望。     

Characterisation of Archaeglobus fulgidus AlkA hypoxanthine DNA glycosylase activity

The AlkA protein from the archaebacterium Archaeglobus fulgidus was characterised with respect to release of hypoxanthine from DNA. The hypoxanthine glycosylase activity had optimal activity at 60°C at pH 5.0. The enzyme released hypoxanthine from substrates with a preference for dI:dGdI:dT>dI:dC>dI:dA. The presence of a mismatch on either side of the dIMP in the substrate reduced excision efficiency of the hypoxanthine residue at neutral pH, while a mismatch on both sides of the dIMP resulted in total loss of excision. Release of hypoxanthine from DNA required a minimum of two bases on the 5′ side and four bases on the 3′ side of the dIMP residue.     

Human 3-methyladenine-DNA glycosylase: effect of sequence context on excision, association with PCNA, and stimulation by AP endonuclease

Human 3-methyladenine-DNA glycosylase (MPG protein) is involved in the base excision repair (BER) pathway responsible mainly for the repair of small DNA base modifications. It initiates BER by recognizing DNA adducts and cleaving the glycosylic bond leaving an abasic site. Here, we explore several of the factors that could influence excision of adducts recognized by MPG, including sequence context, effect of APE1, and interaction with other proteins. To investigate sequence context, we used 13 different 25 bp oligodeoxyribonucleotides containing a unique hypoxanthine residue (Hx) and show that the steady-state specificity of Hx excision by MPG varied by 17-fold. If APE1 protein is used in the reaction for Hx removal by MPG, the steady-state kinetic parameters increase by between fivefold and 27-fold, depending on the oligodeoxyribonucleotide. Since MPG has a role in removing adducts such as 3-methyladenine that block DNA synthesis and there is a potential sequence for proliferating cell nuclear antigen (PCNA) interaction, we hypothesized that MPG protein could interact with PCNA, a protein involved in repair and replication. We demonstrate that PCNA associates with MPG using immunoprecipitation with either purified proteins or whole cell extracts. Moreover, PCNA binds to both APE1 and MPG at different sites, and loading PCNA onto a nicked, closed circular substrate with a unique Hx residue enhances MPG catalyzed excision. These data are consistent with an interaction that facilitates repair by MPG or APE1 by association with PCNA. Thus, PCNA could have a role in short-patch BER as well as in long-patch BER. Overall, the data reported here show how multiple factors contribute to the activity of MPG in cells.     

Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to remove mismatched normal DNA bases.

The repair of T:G mismatches in DNA is key for maintaining bacterial restriction/modification systems and gene silencing in higher eukaryotes. T:G mismatch repair can be initiated by a specific mismatch glycosylase (MIG) that is homologous to the helix-hairpin-helix (HhH) DNA repair enzymes. Here, we present a 2.0 A resolution crystal structure and complementary mutagenesis results for this thermophilic HhH MIG enzyme. The results suggest that MIG distorts the target thymine nucleotide by twisting the thymine base approximately 90 degrees away from its normal anti position within DNA. We propose that functionally significant differences exist in DNA repair enzyme extrahelical nucleotide binding and catalysis that are characteristic of whether the target base is damaged or is a normal base within a mispair. These results explain why pure HhH DNA glycosylases and combined glycosylase/AP lyases cannot be interconverted by simply altering their functional group chemistry, and how broad-specificity DNA glycosylase enzymes may weaken the glycosylic linkage to allow a variety of damaged DNA bases to be excised.     

Characterization of cold-active uracil-DNA glycosylase from Bacillus sp. HJ171 and its use for contamination control in PCR

In this study, the gene encoding Bacillus sp. HJ171 uracil-DNA glycosylase (Bsp HJ171 UDG) was cloned and sequenced. The Bsp HJ171 UDG gene consists of a 738-bp DNA sequence, which encodes for a protein that is 245-amino-acid residues in length. The deduced amino acid sequence of the Bsp HJ171 UDG had a high sequence similarity with other bacterial UDGs. The molecular mass of the protein derived from this amino acid sequence was 27.218 kDa. The Bsp HJ171 UDG gene was expressed under the control of a T7lac promoter in the pTYB1 plasmid in Escherichia coli BL21 (DE3). The expressed enzyme was purified in one step using the Intein Mediated Purification with an Affinity Chitin-binding Tag purification system. The optimal temperature range, pH, NaCl concentration, and KCl concentration of the purified enzyme was 20–25°C, 8.0, 25 and 25 mM, respectively. The half-life of the enzyme at 40°C and 50°C were approximately 131 and 45 s, respectively. These heat-labile characteristics enabled Bsp HJ171 UDG to control carry-over contamination in the polymerase chain reaction product (PCR) without losing the PCR product. G.A. Kim and M.S. Lee contributed equally to this work.     

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.     

Selective base excision repair of DNA damage by the non‐base‐flipping DNA glycosylase AlkC

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT‐like repeat (HLR) fold. AlkD uses a unique non‐base‐flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3‐methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non‐base‐flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin‐like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3‐methylcytosine (3mC) and N1‐methyladenine (1mA), which are also repaired by AlkB‐catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.