Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase

Thomas J. Begley and Richard P. Cunningham1

Department of Biological Sciences, SUNY at Albany, Albany, NY 12222, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum, a member of the endonuclease III family of repair proteins, excises the pyrimidine base from T–G and U–G mismatches. Unlike endonuclease III, it does not cleave the phosphodiester backbone by a ß-elimination reaction. This cleavage event has been attributed to a nucleophilic attack by the conserved Lys120 of endonuclease III on the aldehyde group at C1' of the deoxyribose and subsequent Schiff base formation. The inability of TDG to perform this ß-elimination event appears to be due to the presence of a tyrosine residue at the position equivalent to Lys120 in endonuclease III. The purpose of this work was to investigate the requirements for AP lyase activity. We replaced Tyr126 in TDG with a lysine residue to determine if this replacement would yield an enzyme with an associated AP lyase activity capable of removing a mismatched pyrimidine. We observed that this replacement abolishes the glycosylase activity of TDG but does not affect substrate recognition. It does, however, convert the enzyme into an AP lyase. Chemical trapping assays show that this cleavage proceeds through a Schiff base intermediate and suggest that the amino acid at position 126 interacts with C1' on the deoxyribose sugar.

Keywords: AP lyase/DNA damage/endonuclease III/thymine DNA mismatch glycosylase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Research on DNA glycosylases has revealed a number of different enzymes that recognize and remove numerous forms of pre-mutagenic DNA lesions. These enzymes are responsible for initiating the base excision repair pathway by cleaving the glycosyl bond to create an apurinic/apyrimidinic (AP) site and, in some cases, by cutting the DNA backbone after base removal. The lesions recognized by glycosylases include non-canonical Watson–Crick base pairs and bases altered by oxidation, alkylation and ionizing radiation damage (for a recent review, see David and Williams, 1998Go). Protein engineering studies on DNA glycosylases can generate information on the basis of substrate specificity and also may provide enzymes with novel specificities. The design of new DNA glycosylases is still in its infancy; however, the creation of a novel enzyme based on the framework of uracil glycosylase represents one of the first attempts to engineer new base-specific enzymes (Kavli et al., 1996Go).

Engineering new glycosylases not only requires knowledge about the specific interactions that govern substrate identification, but also about the chemical species that initiate glycosyl bond cleavage. The endonuclease III family of DNA glycosylases provides a unique set of enzymes that could contribute information about residues that affect both specificity and catalysis. This family of DNA repair enzymes encompasses a number of proteins which recognize different substrates, ranging from damaged pyrimidines to normal bases in pre-mutagenic non-Watson–Crick basepairs. Glycosylases belonging to the endonuclease III family remove a damaged or inappropriate base by glycosyl bond cleavage to create an AP site. This reaction has been proposed to be initiated by an activated water or lysine residue serving as the nucleophile used to attack C1'. These two nucleophiles provide a bifurcation in the mechanism of glycosyl bond hydrolysis for members of the endonuclease III family (Dodson et al., 1994Go). The use of lysine as the nucleophile can lead to cleavage of the DNA backbone by a subsequent ß-elimination reaction or, if an activated water has initiated the attack, the AP site will be left intact (Cunningham, 1997Go). Two members of this protein family that differ with respect to substrate specificity and ß-lyase activity are Escherichia coli endonuclease III, which is a glycosylase/AP lyase, and Methanobacterium thermoformicicum (Mth) thymine DNA mismatch glycosylase, which is a pure glycosylase.

Endonuclease III was originally identified by its ability to nick heavily UV or {gamma}-irradiated DNA (Radman, 1976Go; Katcher and Wallace, 1983Go). It has been characterized as a glycosylase active against damaged pyrimidines, with an associated AP lyase activity that cleaves the DNA backbone 3' to the AP site (Demple and Linn, 1980Go; Katcher and Wallace, 1983Go; Breimer and Lindahl, 1985Go; Boorstein et al., 1989Go; Mazumder et al., 1991Go; Dizdaroglu et al., 1993Go; Hatahet et al., 1994Go). Phosphodiester bond cleavage is catalyzed by the formation of a Schiff base, between the amino group of Lys120 and the C1' aldehyde of the deoxyribose residue. This reaction forms a covalent protein–DNA intermediate in the form of an iminium ion. The formation of a neutral enamine followed by a ß-elimination event cleaves the DNA backbone (Bailly and Verly, 1987Go; Kim and Linn, 1988Go; Mazumder et al., 1991Go). This chemistry can be exploited to trap covalently bound enzyme substrate intermediates by the addition of a strong reducing agent. The catalytic mechanism of endonuclease III (Sun et al., 1995Go; Hilbert et al., 1996Go), Fpg protein (Tchou and Grollman, 1995Go; Zharkov et al., 1997Go) from E.coli, endonuclease V from bacteriophage T4 (Latham et al., 1995Go) and OGG1 from Saccharomyces cerevisiae (Nash et al., 1996Go) has been confirmed in this manner. In addition, this type of trapping assay has been used to identify repair proteins with various substrate specificities in other organisms (Hilbert et al., 1996Go; Nash et al., 1996Go; Lu et al., 1997Go).

Thymine DNA mismatch glycosylase (TDG) has been characterized as a glycosylase active against thymine or uracil residues mismatched with a guanine. The enzyme does not exhibit a detectable AP lyase activity (Horst and Fritz, 1996Go). The gene for TDG was initially identified on a plasmid carrying the Mth TI restriction-modification system in the thermophilic archaeon M.thermoformicicum. The modification methylase of this restriction-modification system forms a 5-methylcytosine (5meC) residue at the internal cytosine in a GGCC sequence (Nölling et al., 1992Go). This is an unexpected modification system for a thermophilic organism, because of the high rate of deamination of 5meC at 65°C, the optimal growth temperature of M.thermoformicicum. The formation of thymine–guanine mismatches arising from the deamination of 5meC could be detrimental if left uncorrected, by promoting C–G to T–A transition mutations upon replication. TDG has therefore been implicated in counteracting the mutagenic effects of 5meC deamination by removing the newly formed thymine from a T–G mismatch. TDG also has the ability to remove uracil from U–G mismatches and could be considered a backup uracil glycosylase similar to mismatch-specific uracil glycosylase (MUG) from E.coli (Barrett et al., 1998Go) and TDG from humans (Nedderman and Jiricny, 1994Go; Waters and Swann, 1998Go).

Protein alignment of endonuclease III homologs with TDG (Figure 1Go) shows a number of similarities. A highly conserved C-terminal cysteine ligation pattern associated with the 4Fe–4S cluster of endonuclease III is present in this thermophilic glycosylase. The active site region of the endonuclease III homologs shows completely conserved aspartic acid (D138) and lysine (K120) residues (Thayer et al., 1995Go; Girard et al., 1997Go). The conserved aspartic acid residue is present in TDG (D144), but the lysine residue involved in Schiff base formation by endonuclease III has been replaced by a tyrosine (Y126) residue in TDG. In this study we have attempted to convert the reaction mechanism of TDG from a pure glycosylase to a glycosylase/AP lyase. We have also investigated the binding of TDG to a U–G substrate and the cognate product which contains an AP site opposite a guanine residue (AP-G) and we have examined the catalytic properties of a Y126K mutant form of the enzyme.



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Fig. 1. Protein alignment of endonuclease III homologs. All proteins of the endonuclease III family of DNA glycosylases contain a completely conserved D144 residue. The Y126K mutation of TDG lies near a conserved LPGVG motif that was found to compose part of the helix–hairpin–helix domain in the crystal structure of endonuclease III from E.coli (Thayer et al., 1995Go).

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amino acid alignment

Proteins were aligned using the program ClustalW (Higgins et al., 1996Go). Representative DNA glycosylases of the endonuclease III family from the following organisms, with accession numbers, include TDG from M.thermoformicicum, 232205; a C-terminal truncated version of MutY from E.coli (EcoMutY), 127559; endonuclease III from E.coli(EcoEndoIII), 119328; and a C-terminal truncated version of the ultraviolet-N-glycosylase from Micrococcus luteus (MluUVendo), 2506195. The alignment was processed in Boxshade with a highlight factor of 1.0.

Bacteria and plasmids

E.coli JM109 and BW415 thr-1 leuB6 proA2 hisG4 argE3 thi-1 ara-14 lacY1 galK2 xyl-5 mtl-1 rpsL31 tsx-33 {lambda} supE44{Delta}(manA-nth)84 ksgB1 (endonuclease III deficient) (Weiss and Cunningham, 1985Go) were from our laboratory strain collection. BW415DE3 was prepared using a {lambda}DE3 lysogenization kit from Novagen, Inc. Plasmid pUV2, a pUC plasmid containing ORF10, the gene coding for TDG, derived from pFV1 was a generous gift from Dr J.Nölling (Nölling et al., 1992Go). Plasmid pUC19 was from our laboratory and pET14B was from Novagen. Plasmid pUBS520 containing the dnaY gene was a generous gift from Dr P.Buckel (Brinkman et al., 1989Go).

Preparation and manipulation of DNA

Plasmid DNAs were prepared from the host strain JM109 using a QIAprep spin plasmid kit from Quiagen. A DNA fragment containing ORF10 was removed from pUV2 on a 1.6 kB DraI–EcoRI fragment and ligated into the polylinker site of pUC19 digested with HincII and EcoRI. The TTG start codon of ORF10 was changed to an ATG codon with the introduction of an NcoI site by PCR amplification. A BamHI site was also introduced 3' to the gene at the same time using the primers 5' GTG GGG CTG GAT TTC CAT GGA TGA TGC TAC TAA T 3' and 5' CGA CGG CCA GTG GAT CCA AGG GGG CTG ATG 3'. These new restriction sites were used to clone ORF10 into the plasmid pET14B to create pET14BORF10. The Y126K mutation was prepared using the mega primer mutagenesis method (Barik and Galinski, 1991Go). Both the wild-type and mutant gene were sequenced using the Sequenase v2.0 system from United States Biochemical.

Protein induction and purification

pET14BORF10 and pUBS520 were transformed into E.coli strain BW415DE3. Transformants were selected on tryptone yeast (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter H2O, pH 7.2) (TY) plates containing 50 µg/ml of ampicillin and 34 µg/ml of kanamycin. Cells were grown in TY broth supplemented with 50 µg/ml ampicillin and 34 µg/ml kanamycin at 37°C to an OD595 = 0.5 and induced with 1 mM IPTG for 5 h. The cells were harvested by centrifugation at 17 000 g for 20 min and the cell paste was stored at –80°C. A 15 g amount of cell paste was thawed and suspended in 25 ml of 50 mM KPO4, pH 7.2, 1 M NaCl, 2.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). A beaker containing the cell suspension was placed in an ice slurry and the cell suspension was sonicated five times for 3 min each with a Branson sonifier at maximum power output. The sonicate was stirred at 0°C for 12 h. It was then centrifuged at 48 000 g for 20 min. The supernatant (Fraction I, 23 ml) was applied to a Sephacryl S100 HR (Pharmacia) column (2.6x120 cm). Protein was eluted with 50 mM KPO4, 1 M NaCl, pH 7.2, at 1 ml/min. Fractions containing the product of the ORF10 gene, TDG, were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and the characteristic yellow color associated with the 4Fe–4S cluster and pooled. This mixture was slowly diluted with 50 mM KPO4, pH 7.2, to a final NaCl concentration of 250 mM (Fraction II).

Fraction II (460 ml) was passed through a Q Sepharose Fast Flow (Pharmacia) column (2.6x15 cm) in 50 mM KPO4, pH 7.2, 250 mM NaCl to remove residual chromosomal DNA. The eluate was applied directly to a 5 ml HiTrap Heparin (Pharmacia) column and the column was developed with a 100 ml gradient of 250–1000 mM NaCl at 3 ml/min. TDG eluted at 500 mM NaCl and fractions containing the enzyme were pooled and dialyzed against 50 mM KPO4, pH 7.2 (Fraction III). Fraction III (15 ml) was applied to a single-stranded DNA agarose (Pharmacia) column (1.6x6 cm) at 1 ml/min. The column was washed with 20 ml of 50 mM KPO4, pH 7.2, and developed with a 50 ml gradient from 0 to 1 M NaCl at 1 ml/min. TDG eluted at 1 M NaCl and fractions containing the enzyme were pooled and dialyzed overnight against 1 l of 50 mM KPO4, pH 7.2 (Fraction IV). This pool (46 ml) was applied to a 5 ml HiTrap Heparin (Pharmacia) column. Bound TDG was eluted with 1 M NaCl (Fraction V). This column was run to remove any DNA leached from the DNA agarose column. The fractions containing TDG were determined by SDS–PAGE, glycosylase activity and the characteristic yellow color of the protein containing an Fe–S cluster.

The total protein in each fraction was determined by the method of Bradford using bovine serum albumin (BSA) as a standard (Bradford, 1976Go). The concentration of TDG was calculated using an {varepsilon}410 of 14.4 mM–1 cm–1 for the Fe–S cluster. Mutant versions of TDG were purified using the same protocol.

Spectral determination

The absorption spectrum of TDG (0.66 mg/ml) in 50 mM KPO4, 400 mM NaCl, pH 7.2, was recorded using a Hitachi U-2000 UV/VIS spectrophotometer.

Substrates

Oligonucleotides U (5'-GCAGCGCAGUCAGCCGACG-3'), G (5'-CGTCGGCTGGGCCCTGCGCTGC-3') were purchased from Oligos etc. and were supplied ready to use as lyophilized samples. U-oligonucleotides were labeled with [32P]ATP from New England Nuclear using T4 polynucleotide kinase from New England Biolabs according to the supplier's specifications. Unincorporated [32P]ATP was removed using Sephadex G25 spin columns from Pharmacia. The [32P]U and G oligonucleotides were annealed on ice in a 1:1 ratio to generate a duplex 19-mer oligonucleotide with a U–G mismatch. Thymine glycol and urea containing oligonucleotides were a gift from Dr P.Bolton, Wesleyan University. The oligonucleotides were labeled and annealed as described above. To make labeled oligonucleotides containing AP sites, single-stranded uracil-containing oligonucleotides were labeled with 32P in 70 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)–KOH, pH 7.5, 10 mM MgCl2, 0.1 mM dithiothreitol (DTT) and purified on a Sephadex G25 spin column. The uracil residue was removed by treatment in 70 mM HEPES–KOH, pH 7.5, 10 mM MgCl2, 0.1 mM DTT with 50 ng of uracil glycosylase for 1 h at 37°C. Preparation of unlabeled AP 19-mer was carried out in a similar fashion, with the omission of [32P]ATP. The double-stranded [32P]G-AP 19-mer was created by annealing an unlabeled oligonucleotide containing an AP site to an equimolar concentration of 32P-labeled G 19-mer.

Glycosylase assay

U–G mismatch glycosylase activity was monitored by measuring the alkali induced cleavage of an AP site created by TDG acting on a mismatched substrate. [32P]U–G duplex 19-mer (100 nM) was incubated with indicated amounts of TDG in 50 mM HEPES–KOH, pH 7.5, 100 mM KCl, 10 mM EDTA at 65°C for 30 min. The enzyme was then inactivated by the addition of 5 M urea followed by incubation at 100°C for 5 min. The reaction mixture was then incubated with 0.15 M NaOH for 15 min at 55°C to cleave the AP sites generated by TDG. Loading buffer (80% deionized formamide, 50 mM HEPES–KOH, pH 7.5, 1 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) was added to the sample, which was run on a 20% acrylamide gel (8.0x7.3x0.75 cm) containing 8 M urea for 2 h at 200 V in 50 mM HEPES–KOH, pH 7.5. The gels were dried and exposed to X-ray film. The gels were also quantified on a Betagen 603 ß scope. The observed activity of each sample was determined by measuring the formation of product relative to the total substrate present. The equation A = P/P + S was used to quantitate the activity (A), with P being the amount of product formed and S the amount of substrate unaffected. The activity of Y126K TDG on urea- and thymine-glycol containing oligonucleotides was analyzed as described above.

AP lyase assay

AP lyase activity was monitored by measuring strand cleavage of an oligonucleotide containing an AP site. [32P]AP-G 19-mer substrate (100 nM) was incubated with indicated amounts of wild-type, Y126K or Y126S TDG in 50 mM HEPES–KOH, pH 7.5, 100 mM KCl and 10 mM EDTA at 65°C for 30 min. Owing to the non-protein induced cleavage of AP sites observed during urea denaturation at high temperature, these steps were omitted from this assay. Loading buffer was added to samples and they were run on a 20% acrylamide gel (8.0x7.3 cm) containing 8 M urea for 2 h at 200 V in 50 mM HEPES–KOH, pH 7.5. The gels were analyzed on a Betagen 603 ß scope.

Determination of binding constants

Each reaction contained 47 nM 32P end-labeled U–G or AP-G 19-mer, 0–45 µM TDG or Y126K TDG, 50 mM 2-(cyclohexylamino)ethanesulfonic acid (CHES)–KOH, pH 9.2, 100 mM KCl, 10 mM EDTA, 4760 nM cold U–A 19-mer and BSA (1 µg/µl). Reaction mixtures were incubated at room temperature for 30 min prior to loading on an 8% polyacrylamide (19:1) gel (8.0x7.3x1.0 cm) containing 50 mM CHES–KOH, pH 9.2. The gels were run at 150 V in 50 mM CHES–KOH, pH 9.2, for 35 min. The radioactivity of free and complexed DNA was quantitated on a Betagen ß scope. The ratio of free DNA to the total amount of DNA was plotted against the enzyme concentration to obtain binding constants.

NaCNBH3 cross-linking assay

All samples contained 50 mM HEPES–KOH, pH 7.2, 200 mM KCl, 10 mM EDTA, 4.2 µM WT or Y126K TDG. Indicated samples contained 40 mM NaCNBH3 and 42 µM [32P]G-AP 19-mer. Reaction mixtures were incubated at 65°C for 15 min, boiled in 2x SDS–PAGE loading buffer for 5 min and applied to a 12% SDS–PAGE gel. Gels were run at 150 V for 2 h, stained with Coomassie Brilliant Blue, destained, dried and exposed to X-ray film.


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Protein induction and purification

The gene coding for TDG contains 13 arginine codons (AGA, AGG) that are rarely used in E.coli. Because a limiting amount of the cognate tRNA could reduce the translation of the TDG mRNA in E.coli, we expressed the gene for this rare arginine tRNA (dnaY) along with the gene for TDG in an attempt to increase the yield of TDG. Figure 2Go shows that the level of expression of TDG is higher in the presence of this tRNA which is expressed from the plasmid pUBS520. A summary of a typical purification of TDG is provided in Table IGo. This purification yielded 19.5 mg of purified TDG starting from 15 g of cell paste.



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Fig. 2. Coomassie Brilliant Blue-stained SDS–PAGE analysis of cell lysates. Strain BW415DE3 containing pET14bORF10 (lanes 1 and 2) or pET14BORF10 and pUBS520 together (lanes 3 and 4), in the absence (lanes 1 and 3) or presence of 1 mM IPTG (lanes 2 and 4). Lane 5 contains purified TDG as a marker.

 

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Table I. Purification of TDG
 
Spectroscopic analysis

Analysis of TDG by UV/VIS spectrophotometry (Figure 3Go) showed a shoulder at 410 nm, indicative of the presence of an Fe–S cluster (Asahara et al., 1989Go). Using an {varepsilon}410 of 14.4 mM–1 cm–1 for the Fe–S cluster and an {varepsilon}280 of 48.35 mM–1cm–1 for TDG, a theoretical A410/A280 = 0.298 for pure TDG was determined. The value of 48.35 mM–1 cm–1 was calculated assuming a contribution of 36.0 at 280 nm from the protein (ProtParam from ExPASy, Geneva University Hospital and University of Geneva, Geneva, Switzerland), with the addition of 12.35 to account for the absorbance of the Fe–S cluster at 280 nM. The ProtParam program calculates the molar extinction coefficient of a protein based on the number of tyrosine, tryptophan and cysteine residues in the protein (Gill and von Hippel, 1989Go). An experimental A410/A280 value of 0.287 for our highly purified TDG was obtained. The chromophore responsible for the A410 peak in endonuclease III is a 4Fe–4S cluster (Cunningham et al., 1989Go). This cluster is liganded by a characteristic cysteine pattern seen in the subset of DNA repair enzymes including endonuclease III and MutY (Thayer et al., 1995Go). We predict the presence of a 4Fe–4S cluster in TDG based on this homologous cysteine ligation pattern (Figure 1Go) and the absorption spectrum of the protein (Figure 3Go).



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Fig. 3. Spectral analysis of a 0.66 mg/ml solution of TDG.

 
Glycosylase activity

The initial characterization of TDG (Horst and Fritz, 1996Go) showed that the enzyme removed both thymine and uracil mismatched with guanine and that it had no apparent strand cleavage activity. Analysis of TDG under our assay conditions, using a labeled [32P]U–G mismatch, gave results in agreement with Horst and Fritz. At concentrations of 1, 2 and 4 µM of wild-type TDG, over 90% of the substrate was converted to alkali labile products with increased mobility on a 20% denaturing acrylamide gel (lanes 2–4, Figure 4Go). Cleavage of the phosphodiester bond was not observed when the alkali treatment was omitted (data not shown). In contrast, under the same conditions, the Y126K mutant had no observable activity on this U–G substrate (lanes 5–7, Figure 4Go). Since we had altered TDG to resemble endonuclease III, we asked if Y126K TDG could remove either thymine glycol or urea, both substrates of endonuclease III, from a duplex oligonucleotide. The mutant form of TDG also showed no glycosylase activity on these substrates (data not shown).



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Fig. 4. Glycosylase activity of TDG and Y126K against 100 nM [32P]U–G oligonucleotide. Lanes 1–7 are, in order, no protein, 1, 2 and 4 µM TDG and 1, 2 and 4 µM Y126K.

 
Determination of binding constants

We performed quantitative band shift assays to determine the binding constants and binding specificities of TDG and Y126K TDG. Initial studies were performed using the wild-type protein and a [32P]U–G 19-mer (Figure 5Go). The data obtained were fitted to a Langmuir isotherm that is characteristic of single-site, non-cooperative binding (Brenowitz et al., 1986Go; Carey, 1991Go). Assuming a Kd of 9 µM, we calculated a theoretical isotherm for this binding model which closely resembles the experimental data (Figure 6Go). An aliquot of each binding reaction was analyzed for strand cleavage by the addition of 0.2 M NaOH followed by denaturing gel electrophoresis (not shown). This analysis indicated that the formation of AP sites in the substrate increased with TDG concentration; 50% of the total substrate in the 44 µM sample containing TDG contained an AP site. Since the binding data can be fitted to a single isotherm, the binding constant of protein to substrate and product must be very similar. The specificity of the enzyme for the mismatched substrate was tested by removing the 100-fold excess of a U–A 19-mer. The binding constants with and without this competitor were very similar, 7 µM without (not shown) and 9 µM with competitor, indicating that TDG has a high specificity for the U–G mismatch.



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Fig. 5. Gel retardation experiments with wild-type TDG and a [32P]U–G 19-mer.

 


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Fig. 6. Comparison of the model for single-site, non-cooperative binding (solid line) with the experimental values (open squares). The theoretical Langmuir isotherm for TDG was generated using a Kd of 9.6 µM in the equation

where Keq = 1/Kd and Y is the fraction of bound target.

 
We measured the binding of TDG to an oligonucleotide containing a central AP site (Figure 7Go). The data were similar to those seen for a U–G duplex, with 50% binding between 6 and 18 µM. The binding to product was expected since we could model a single isotherm to data obtained from an experiment in which protein was clearly binding to both substrate and product. Affinity for product has been seen previously for proteins involved in base excision repair (Schärer et al., 1996; Parikh et al., 1998Go; Waters and Swann, 1998Go).



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Fig. 7. Gel retardation experiments with wild-type TDG and [32P]AP-G 19-mer.

 
The results of binding studies carried out with Y126K TDG and a U–G substrate are shown in Figure 8Go. This analysis shows a similar result to that seen with wild-type enzyme, with a binding transition between 6 and 18 µM. The ability of this mutant enzyme to bind a U–G substrate with near wild-type affinity indicates that the effect of the Y126K mutation is to block the catalytic activity of TDG, without significantly affecting binding.



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Fig. 8. Gel retardation experiments with TDG Y126K and [32P]U–G 19-mer.

 
Strand cleavage activity

The ability of wild-type TDG to bind AP sites suggested that the presence of a lysine at position 126 could produce an efficient lyase activity in Y126K through the formation of a Schiff base and subsequent ß-elimination 3' to the AP site. We assayed TDG and the Y126K mutant TDG for AP lyase activity (Figure 9Go) and found that the mutant enzyme had acquired this activity, which the wild-type enzyme lacks. A Y126S TDG mutant was also made and tested for AP lyase activity. This was done to address the question of whether lysine at position 126 was responsible for the AP lyase activity or if the Y126K alteration allowed for a neighboring lysine (Lys125) to have accessibility to the sugar moiety. The Y126S mutant has no detectable lyase activity (data not shown), supporting our contention that a lysine at position 126 is responsible for the new AP lyase activity of Y126K TDG.



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Fig. 9. AP lyase activity of wild-type TDG and TDG Y126K. The substrate was 100 nM [32P]AP-G 19-mer. The assay was performed as stated previously with the omission of NaOH. Lanes 1–8 have increasing concentrations of either WT TDG (A) or TDG Y126K (B) (0, 0, 0.02, 0.2, 0.4, 0.8, 1.6 and 3.2 µM).

 
Reductive cross-linking

All known naturally occurring AP lyases function via Schiff base formation. We analyzed the cleavage reaction catalyzed by Y126K TDG to confirm that it used a similar chemistry. NaCNBH3 reductive cross-linking was used to trap potential TDG–substrate intermediates. Enzyme and a duplex 19-mer containing an AP site radioactively labeled on the complementary strand were cross-linked and then displayed by SDS–PAGE (Figure 10Go). Wild-type (lanes 1–3) and Y126K TDG (lanes 4–6) were assayed in this manner. The gels were stained with Coomassie Brilliant Blue, destained, dried and analyzed by autoradiography. No shift of stained proteins or radioactively labeled bands was observed with wild-type protein. This was not the case for the Y126K enzyme. There is a distinct shift in the apparent molecular weight of the enzyme as seen in lane 6, which contains Y126K in the presence of substrate and NaCNBH3. Analysis of the possible products of a covalent enzyme–DNA intermediate (Figure 11Go) shows that a complex can be trapped either before or after the cleavage event has occurred. A single-stranded 19-mer oligonucleotide has a theoretical molecular weight of 6.3 kDa, while the cleaved product has a molecular weight of ~3 kDa. Analysis of the Coomassie-stained products in lane 6 reveals uncomplexed Y126K TDG which ran at 25 kDa. Three additional bands which ran at 28 kDa (A), 31 kDa (B) and 35 kDa (C) were also observed. The 28 kDa band represents a cleaved oligonucleotide bound to TDG (3 + 25.4 kDa). The 31 kDa band (B) corresponds to an intact single-stranded oligonucleotide bound to TDG (25.4 + 6). The 36 kDa band (C) corresponds to an intact double-stranded oligonucleotide cross-linked to TDG (25.4 + 12). A radioactive band corresponding to the 36 kDa complex was observed by autoradiography. The labeling of this 36 kDa band confirms that it contains TDG cross-linked to the double stranded oligonucleotide because only the strand complementary to the oligonucleotide containing the AP site was labeled. This experiment shows that covalent protein–DNA intermediates can be trapped under reducing conditions and confirms that the reaction mechanism proceeds through a Schiff base intermediate.



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Fig. 10. Reductive cross-linking assay performed with wild-type TDG and TDG Y126K. Lanes 1–3 contain wild-type TDG and lanes 4–6 contain TDG Y126K. Lanes 2 and 5 contain substrate oligonucleotide. Lanes 3 and 6 contain substrate and NaCNBH3. Bands marked A, B and C correspond to molecular weights of 28, 31 and 35 kDa, respectively, with C also corresponding to a radioactive band on an autoradiograph (not shown).

 


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Fig. 11. The proposed reaction mechanism of endonuclease III and Y126K TDG acting upon the open-chain aldehyde form of an AP site (A). An iminium ion is formed from the catalytic lysine and the open-chain aldehyde of the abasic site. The base-catalyzed formation of an enamine precedes cleavage of the DNA backbone and the formation of an {alpha},ß-unsaturated aldehyde. NaCNBH3 treatment of this reaction can trap protein–substrate complexes at two points during this ß-elimination reaction. This can occur before (B) or after (C) strand cleavage.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The endonuclease III family of DNA repair enzymes includes members which are pure glycosylases and those which are glycosylase/AP lysases. Representatives include endonuclease III, UV-endonuclease, MutY and TDG. We have previously shown that Lys120 in endonuclease III is the active site for the AP lyase and glycosylase activities (Thayer et al., 1995Go) and that mutant enzymes which lack this residue cannot be reductively cross-linked to oligonucleotides containing an AP site (D.Xing and R.P.Cunningham, unpublished results). Others have shown that the homologous lysine plays the same role in yOGG1 and hOGG1 (Nash et al., 1996Go; Lu et al., 1997Go), which are also related to endonuclease III. MutY and TDG, which remove adenine and thymine from A–G and T–G mismatches, respectively, have either a serine or a tyrosine at the equivalent position and appear to be pure glycosylases, although there are several reports in the literature suggesting that MutY may have an associated lyase activity (Lu et al., 1992Go; Manual and Lloyd, 1997Go). We suggest that this MutY lyase activity may be an adventitious activity resulting from attack by a nearby lysine on preformed AP sites and is not directly associated with the primary cleavage of the glycosyl bond. MutY and TDG remove normal bases from mismatched DNA base pairs. Normal DNA bases can be hydrolyzed at an increased rate under acidic conditions, with purines being far more labile than pyrimidines, suggesting that protonation could play a role in the activation of undamaged purines and pyrimidines for hydrolysis. Endonuclease III, on the other hand, removes oxidized pyrimidines which have relatively stable glycosyl bonds under acidic conditions. Mechanistically, it would appear that MutY and TDG need to activate adenine or thymine as a leaving group by protonation. An activated water molecule could then attack the glycosyl bond. Endonuclease III, on the other hand, uses Lys120 as the attacking nucleophile. This results in an iminium ion intermediate which ultimately leads to a ß-elimination event and cleavage of the phosphodiester backbone.

We have confirmed that wild-type TDG is a pure glycosylase by showing that there is no detectable cleavage of the phosphodiester backbone after base release and that there are no intermediates in the course of the enzymatic reaction that can be trapped with sodium cyanoborohydride. We then made the Y126K TDG to see if we could convert a pure glycosylase to a glycosylase/AP lyase by replacing tyrosine with lysine. This interconversion of reaction mechanisms presupposes that Tyr126 is responsible for activating or aligning a water molecule for a hydrolytic attack on the glycosyl bond. Unexpectedly, we found that a Y126K mutant of TDG was deficient in glycosylase activity on an oligonucleotide containing a U–G mismatch.

We then investigated whether the Y126K TDG mutant could still recognize and bind an oligonucleotide containing a U–G mismatch using gel retardation assays. Because we showed that the Y126K TDG binds as tightly and as specifically to a U–G mismatch as does the wild-type TDG, we then asked whether Lys126 would be able to catalyze a ß-elimination event at an AP site. The mutant enzyme was able to carry out this reaction, which indicates that the lysine residue at position 126, which we introduced into the enzyme, was able to initiate a nucleophilic attack on the C1' position of the aldehyde of the AP site, leading to a ß-elimination event. We confirmed this mode of action by showing that the Y126K mutant enzyme can be reductively cross-linked to an oligonucleotide containing an AP site substrate by sodium cyanoborohydride.

Our results suggest that the tyrosine residue at position 126 in wild-type TDG could play a role in directing a nucleophilic attack by water on the scissile glycosyl bond. The presence of a lysine at this position in the mutant enzyme could inhibit the proper positioning of an activated water or could displace this nucleophile from the active site. The inability of the lysine residue itself to act as the nucleophile could be explained by differences in the chemical reactivity of normal and damaged nucleotides. Work done on transition state analogs of the DNA glycsoylases has begun to provide insights into this problem. Studies have shown that endonuclease III, which uses a lysine as a nucleophile, specifically binds the transition state mimic, pyrrolidine, whereas MutY, which uses a water as a nucleophile, will not differentiate it from a tetrahydrofuran moiety (Schärer et al., 1998Go). We have shown that lysine at position 126 is catalytically competent for AP lyase activity, so that loss of glycosylase activity cannot be due to gross structural alterations at the active site.

We have observed that TDG seems to bind with equal affinity and specificity to both a substrate oligonucleotide containing a U–G mismatch and product oligonucleotide containing an AP site opposite the orphan guanine residue. This tight binding to product could act to reduce enzyme activity considerably. We have found that TDG does not exhibit robust activity under steady-state conditions (Begley,T.J., Haas,B.J. and Cunningham,R.P. in preparation) and other reports suggest that MutY also has a very low activity (Bulychev et al., 1996Go; Porello et al., 1998aGo,bGo). We are currently investigating the affect of product inhibition on TDG.

Further studies on the various members of the endonuclease III family are required to determine the basis of substrate specificity and requirements for catalytic activity. Our preliminary results with TDG suggest that glycosylase and glycosylase/AP lyases cannot be interconverted by simple amino acid changes. We have also observed that product binding is very tight. Since the base that is actually removed by enzyme action does not seem to play a role in the energetics of binding, we must look for other determinants of substrate specificity used by TDG.


    Acknowledgments
 
We thank Don Orokos, Tony Demarko and Eric Boneventure for help in preparing the figures. We also thank Dr Philip Bolton for generously providing the urea- and thymine glycol-containing oligonucleotides. This work was supported by NIH grant GM46312.


    Notes
 
1 To whom correspondence should be addressed. E-mail: moose{at}csc.albany.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received September 15, 1998; revised December 22, 1998; accepted December 28, 1998.