©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanisms of DNA Demethylation in Chicken Embryos
PURIFICATION AND PROPERTIES OF A 5-METHYLCYTOSINE-DNA GLYCOSYLASE (*)

(Received for publication, November 28, 1994; and in revised form, February 14, 1995)

Jean-Pierre Jost (§) Michel Siegmann Lijie Sun Roland Leung

From the Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously shown that in developing chicken embryos and differentiating mouse myoblasts, the demethylation of 5-metCpGs occurs through the replacement of 5-methylcytosine by cytosine (Jost, J. P.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4685-4688; Jost, J. P. & Jost, Y. C. (1994) J. Biol. Chem. 269, 10040-10043). We have now purified over 30,000-fold a 5-methylcytosine-DNA glycosylase from 12-day-old chicken embryos. The enzyme copurifies with a mismatch-specific thymine-DNA glycosylase and an apyrimidic-endonuclease. The reaction product of the highly purified 5-methylcytosine-DNA glycosylase is 5-methylcytosine. The copurified apyrimidic-endonuclease activity cleaves 3` from the apyrimidic sugar. A 52.5-kDa peptide, isolated as a single band from preparative SDS-polyacrylamide gels, has both the 5-methylcytosine-DNA glycosylase and the mismatch-specific thymine-DNA glycosylase activities. 5-Methylcytosine-DNA glycosylase has an apparent pI of 5.5-7.5 and maximal activity between pH 6.5 and 7.5. The K for hemimethylated oligonucleotide substrate is 8 times 10M with a V(max) of 4 times 10 mol/h/µg protein. 5-Methylcytosine-DNA glycosylase binds equally well to methylated and non-methylated DNA. The enzyme reacts six times faster with the hemimethylated DNA than with the same bifilarly methylated DNA sequence, and single-stranded methylated DNA is not a substrate. The action of the enzyme is distributive.


INTRODUCTION

Through DNA methylation, it is possible to change the information content of DNA that can affect differentiation and development(1, 2, 3) . The specific DNA methylation pattern results from the combination of maintenance/de novo methylation, demodification of the methylated CpGs, by sequence-specific DNA binding proteins (transcription factors)(1, 2, 3) , and possibly by cis-acting DNA elements (4, 5) . Targeted disruption of the DNA methyltransferase gene in mice has given strong evidence for the pivotal role of DNA methylation during embryonic development(6) . Active enzymatic demodification of methylated CpGs may be operational in several systems and may also play a crucial role during differentiation and development ( (7) and references therein). Recently, we have shown that a nuclear cell-free system from chicken embryos can promote the active demethylation of DNA (8) . One enzyme, coined 5-methyl-CpG endonuclease, has no sequence specificity and cleaves only the methylated CpGs. A similar enzymatic activity was also observed in differentiating mouse myoblasts (7) and in preimplantation and postimplantation mouse embryos.^1 More recently, Vairapandi and Duker (9) have identified in HeLa cell lysate a 5-methylcytosine-DNA glycosylase, which may be also involved in the active demodification of DNA.

While purifying the 5-methyl-CpG endonuclease, we discovered that this enzymatic activity is a combination of 5-methylcytosine-DNA glycosylase and AP(^2)-endonuclease. In addition, 5-methylcytosine-DNA glycosylase copurifies with a mismatch-specific thymine-DNA glycosylase.


MATERIALS AND METHODS

Preparation of Crude Nuclear Extracts (in the Cold Room)

12-day-old chicken embryos were used throughout these experiments. Batches of 60 embryos (about 300 g, wet weight) were processed at a time. Embryos were thoroughly washed with ice-cold 0.15 M NaCl and homogenized with a Waring blender in 200 ml of ice-cold solution A (10 mM Hepes, pH 7.5, 100 mM KCl, 3 mM MgCl(2), 0.1 mM EDTA, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 2 mM dithiothreitol (DTT), 0.5 mM spermidine, 0.15 mM spermine). Further homogenization was carried out with a glass-Teflon homogenizer (7 strokes at 1500 rpm). The crude nuclei were sedimented at 1000 times g at 0 °C. Sediments were once more homogenized in buffer A as indicated above. Upon centrifugation, the sediments were resuspended by homogenization in a total volume of 200 ml of buffer A. Nuclei were lysed by the slow addition of 20 ml of 4 M ammonium sulfate. After 30 min in ice, the viscous mass was centrifuged at 140,000 times g for 90 min at 2 °C. The clear supernatant fraction (130 ml) was collected and stored in aliquots at -80 °C.

Chromatography on Heparin-Sepharose (FPLC at Room Temperature)

Crude nuclear extracts were dialyzed at 4 °C with one change of buffer for 4 h against 20 samples volumes of buffer B (10 mM Hepes, pH 7.5, 0.1 M KCl, 5 mM EDTA, 10% (v/v) glycerol, 2 mM benzamidine, 1 mM DTT). Dialyzed samples were centrifuged at 16,000 times g/10 min to remov e insoluble proteins. A column of Heparin-Sepharose (10 ml) equilibrated in buffer B was loaded with 100-140 mg of dialyzed post-nuclear solutions at a speed of 2 ml/min. Elution was carried out at 10 ml/min with a KCl step gradient in buffer B. Most of the activity was eluted with 0.4 and 0.5 M KCl. Active fractions were precipitated with solid ammonium sulfate to 60% saturation. After 30 min in ice, the precipitate was collected by centrifugation at 100,000 times g for 1 h. Sediments were stored at -80 °C.

Chromatography on DEAE and CM-Sepharose FPLC (in the Cold Room)

Above sediments were dissolved in a minimal amount of buffer C (20 mM Hepes, pH 7.5, 10 mM EDTA, 100 mM NaCl, 10% glycerol, 1 mM benzamidine, 1 mM DTT) and dialyzed for 3 h against 1 liter of the same buffer. For one column of 1 ml of DEAE-Sepharose, we routinely loaded 15-20 mg of protein. The flowthrough containing 5-methylcytosine-DNA glycosylase was directly loaded onto a 2-ml column of CM-Sepharose. The CM-Sepharose column was sequentially eluted with buffer C containing 0.1, 0.2, and 0.3 M, NaCl and the enzyme was eluted with a linear gradient of 0.3-0.5 M NaCl at a flow rate of 2 ml/min. Most enzyme activity eluted between 0.35 and 0.5 M NaCl. Active fractions were pooled and precipitated with ammonium sulfate to 70% saturation. Protein sediments were kept at -80 °C.

Affinity Chromatography on DNA Dynabeads (on Ice)

Protein sediments were dissolved in a minimal amount of buffer C and dialyzed for 3 h against 500 ml of the same buffer. Upon centrifugation, 400 µg of the dialyzed protein in 300 µl of buffer were mixed with the affinity matrix in an Eppendorf tube. 40 ng of Escherichia coli DNA were added per µg of protein as nonspecific competitor. Beads were maintained in suspension by gentle stirring. After 30 min of incubation, beads were sedimented, and the supernatant fraction was removed. Beads were washed four times with 1 ml of dialysis buffer. Proteins were eluted with 2 times 60 µl of 1 M NaCl in dialysis buffer. Protein concentration was determined by the Bradford reaction (Bio-Rad). The same degree of purity could be obtained by chromatography on a Mono-S column where the 5-methylcytosine-DNA glycosylase eluted with 0.3-0.4 M NaCl. Chromatography on DNA containing a G/T mismatch was carried out as described by Neddermann and Jiricny(10) .

Preparative SDS-Polyacrylamide Gel Electrophoresis (in the Cold Room)

Separation was performed on a 10 or 12% polyacrylamide gel (acrylamide:bisacrylamide, 29:1) with a 5% stacking gel(11) . Dimensions of the gel were 150 times 200 times 1 mm. Per lane, 20 µg of protein (post affinity chromatography) were separated. Proteins were first treated for 15 min at 37 °C in 20 mM Hepes, pH 7.0, 2% SDS, 20% glycerol, 2% mercaptoethanol, and 0.03% bromphenol blue. Electrophoresis was carried out for 4 h at 250 V (constant voltage).

Bands in the gel were visualized by the CuCl(2) staining procedure, cut out, and destained(12) . Proteins were extracted and denatured by the guanidium hydrochloride procedure as described by Hager and Burges(13) . The relative amount of protein per band was determined by comparison with a silver-stained dilution series of specific protein standards.

Liquid-phase isoelectrofocusing was carried out in a mini-Rotofor cell (Bio-Rad) for 4 h at 4 °C at 8 watts(14) . After electrofocusing, 20 fractions were collected, and the pH was measured. Samples were dialyzed for 3 h against 500 ml of 20 mM Hepes, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1 mM benzamidine, 1 mM DTT, and 10% glycerol (v/v). Activity of 5-methylcytosine-DNA glycosylase was determined as outlined below.

Enzyme Assays

For convenience, we used the assay of specific cleavage at the mCpGs as previously described(7, 8) . This assay detects the combined action of 5-methylcytosine-DNA glycosylase and AP-endonuclease. At a later stage of purification, when the AP-endonuclease was separated from the 5-methylcytosine-DNA glycosylase, it was necessary to initiate a break at the apyrimidic site by subjecting the reaction mixture to alkaline hydrolysis (0.1 M NaOH at 90 °C for 30 min). Mismatch-specific thymine-DNA glycosylase was assayed with oligonucleotides F or G (Table 1). The assays were carried out in a total volume of 25-50 µl containing 25 mM Hepes, pH 7.5, 20 mM EDTA, 0.1 mM ZnCl(2), 1 mM DTT, 0.05 mM ATP, 50 µg of bovine serum albumin (enzyme grade), and 20 ng of the appropriate end-labeled oligonucleotide (see Table 1) of a specific radioactivity of 35,000 cpm/ng. Upon addition of the protein fraction (1 ng-10 µg), incubation was carried out for 1 h at 37 °C. Samples were then diluted to 150 µl with 0.1% SDS in H(2)O and directly extracted with phenol chloroform and ethanol precipitated. The sediments were either directly dissolved in formamide dye(11) , denatured, and analyzed on a 20% polyacrylamide sequencing gel or they were subjected to alkaline hydrolysis in 6 µl of 0.1 M NaOH at 90 °C for 30 min. Specificity of the alkaline hydrolysis was tested in duplicate samples, treated under the same conditions in 6 µl of Hepes/HCl, pH 6.6. Upon hydrolysis, samples were mixed with 6 µl of formamide dye, heated 3 min at 95 °C, and loaded onto the preheated sequencing gel. As a size marker, we used the labeled oligonucleotide B (Table 1) cleaved with MspI. Where indicated, the specific band of reaction product was excised from the sequencing gel and counted for radioactivity. Values of parallel controls were subtracted from test values. As a further assay for the 5-methylcytosine-DNA glycosylase, we used hemimethylated oligonucleotide tritium labeled at the 5-methylcytosine. The DNA substrate pBR322 was methylated with HpaII methylase in the presence of S-adenosyl-L-[methyl-^3H]methionine (100 µCi for 20 µg of plasmid DNA). Labeled DNA was cleaved with EcoRI, mixed with an equal amount of EcoRI cleaved non-labeled pBR322 DNA, heat denatured, and slowly renatured. Upon ethanol precipitation, the labeled hemimethylated DNA was dissolved into 100 µl of 50 mM EDTA. The reaction product of the 5-methylcytosine-DNA glycosylase assay was analyzed by thin layer chromatography (TLC). 5-Methylcytidine and 5-methylcytosine were separated on TLC plastic sheets coated with 0.2 mm of silicagel 600 F254. The solvent system was chloroform:methanol (80:20 (v/v)). Spots corresponding to 5-methylcytidine and 5-methylcytosine (10-µg carrier) were visualized under UV light, cut out, and counted in scintillation fluid. 5-Methyl-dCMP was separated from dTMP and dCMP by using TLC silicagel plates developed with 4 M ammonium sulfate, isopropanol, 1 M sodium acetate, pH 5 (80:5:15). Guanosine 5`-monophosphate was separated from the other nucleotide monophosphate on TLC plastic sheets coated with 0.1 mm of cellulose MN 300 polyethylenimine. Plates were first treated for 30 min with 50% methanol in water with 0.01% Triton. Upon drying, plates were loaded and developed with 1.5 M KH(2)PO(4), pH 3.4.



Analysis of 5` Termini of Cleaved Oligonucleotides

Identification of the terminal nucleotide of the DNA fragment cut by the combined action of 5-methylcytosine-DNA glycosylase and AP-endonuclease was carried out with the modified version of the method of Cedar et al.(15) as described by Bestor et al.(16) .

Gel Mobility Shift Assay and UV Cross-linking

End-labeled oligonucleotides were incubated with the protein fraction for 1 h at 37 °C as for the standard assay of methylcytosine-DNA glycosylase in the presence of E. coli DNA as a nonspecific competitor. The reaction product was separated on a 5% native polyacrylamide gel (acrylamide:bisacrylamide, 29:1) prepared in 0.25 times TBE (10 times TBE is 0.89 M Tris base, 0.89 M boric acid, 0.02 M EDTA, pH 8.3). Separation was carried out at room temperature at constant amperage (30 mA) in 0.25 times TBE. Upon autoradiography, bands were cut out of the gel, and protein was eluted by crushing the piece of gel in the assay buffer. Enzyme activity was directly measured by adding to the crushed gel an excess of the labeled DNA substrate and incubating it at 37 °C for 2 h. The reaction mixture was then extracted with phenol and chloroform as indicated above.

For UV-cross-linking experiments, oligonucleotide D had in the vicinity of the mCpG on the opposite strand two bromodeoxyuridine and one adenosine labeled at high specific activity. Upon formation of the complex, the reaction mixture was separated by gel shift in the dark. Band ``a'' was UV irradiated for 10 min with a transilluminator, and upon extraction and DNase I treatment, the residual protein-DNA complex was separated on a 10% SDS-polyacrylamide gel.

Synthesis of Oligonucleotides and Affinity Matrix

Oligonucleotides methylated at the CpG or non-methylated were synthesized by using an Applied Biosystems model 380 A synthesizer and purified by electrophoresis on 8 or 10% polyacrylamide urea gels. All oligonucleotides used in the present work are listed in Table 1. 5`-ends were labeled with T4 polynucleotide kinase and [-P]ATP, while 3`-ends were labeled by filling in with sequenase version 2, according to the protocol of the manufacturer. Biotinylated double-stranded DNA was linked to Dynabeads according to the indications of the manufacturer.

Chemicals and Enzymes

Benzamidine was purchased from Fluka AG (Buchs/SG, Switzerland). Phenylmethylsulfonyl fluoride was from Boehringer Manheim, and polynucleotide kinase and restriction enzymes were from Biofinex (Praroman, CH-1724 Switzerland). Heparin-Sepharose CL-6B, DEAE-Sepharose, and CM-Sepharose fast flow were obtained from Pharmacia Biotech Inc. Dynabeads-streptavidin was purchased from Milan Analytica AG (CH-1634 LaRoche, Switzerland), and Collodion dialysis bags were from Sartorius AG (D-3400 Göttingen, Germany). TLC cellulose polyethylenimine plates were obtained from Machery-Nagel Co. (D-516 Düren, Germany), and TLC plastic sheets coated with silica gel were from Merck; S-adenosyl-L-[methyl-^3H]methionine (71 Ci/mmol), [alpha-P]dATP, and [-P]ATP triethylammonium salt (3000 Ci/mmol) were purchased from Amersham Corp. Sequenase version 2 was from U. S. Biochemical Corp.


RESULTS

Purification of 5-Methylcytosine-DNA Glycosylase

5-Methylcytosine-DNA glycosylase was purified from 12-day-old chicken embryos by a combination of five different steps as outlined in detail under ``Materials and Methods.'' A summary of the purification is given in Table 2. For a 30,000-fold purification, we had a recovery of only 0.27% of the total initial activity. Big losses of cleavage activity was always observed upon the separation of the proteins on the SDS-polyacrylamide gels and its subsequent extraction, denaturation, and renaturation steps. Part of the losses of activity was also due to the separation of AP-endonuclease activity from the methyl-cytosine-DNA glycosylase. This is clear in Fig. 1(middlepanel, compare lanes1 and 2), where an alkaline hydrolysis of the apyrimidic sugar resulted in the ``recovery'' of specific cleaved DNA fragment. 5-Methylcytosine-DNA glycosylase could be stabilized by the addition of 10% glycerol, 1 mM DTT to the buffers, and by serum albumin (50 µg/50 µl) added to the reaction mixture (Fig. 5, compare fullcircles and fullsquares). 5-Methylcytosine-DNA glycosylase copurifies with the mismatch-specific thymine-DNA glycosylase, and the ratio of the two enzymes remained constant (see Table 2, columnf) throughout the purification ( Table 2and Fig. 1(middlepanel) and 2).




Figure 1: Analysis of the reaction product (20% urea polyacrylamide sequencing gel) of the 5-methylcytosine-DNA glycosylase (assay with oligonucleotide A) and the mismatch-specific thymine-DNA glycosylase (assay with oligonucleotide G) purified on Mono-S (upper panel) and from the 52.5-kDa peptide eluted from 10% SDS-polyacrylamide preparative gel (middle panel). The lowerpanel shows the cleavage product obtained with a fraction from Mono-S, incubated with the oligonucleotide F (Table 1), containing on the same strand a G/T mismatch and a methylated CpG (methylcytosine (mC) + G/T). Cleavage at the G/T mismatch and 5 met-C gives, respectively, 26- and 29-base-long-labeled oligonucleotides. G/T of the lowerpanel is the incubation of the same enzyme preparation with the oligonucleotide G (Table 1) labeled on the upperstrand (strand containing the mismatched G). For lanes1 and 5, the reaction product was heated for 30 min at 90 °C in Hepes-HCl, pH 6.6, whereas for lanes2 and 6, the reaction product was incubated for 30 min at 90 °C in 0.1 M NaOH. Lanes3 and 4 are the corresponding controls to 1 and 2 incubated without protein, and lanes7 and 8 are the controls of lanes5 a nd 6 incubated without protein.




Figure 5: Kinetics of the cleavage reaction with hemimethylated oligonucleotide A and the bifilarly methylated oligonucleotide B. Fullcircles show the kinetics of the reaction at a specific CpG site in the presence of 50 µg of bovine serum albumin and 2 µg of protein fraction eluted from the Mono-S column. Fullsquares show the same reaction mixture incubated in the absence of bovine serum albumin. The opensquares and opencircles are the product of reaction obtained with bifilarly methylated DNA in the presence of 2 µg of the same enzyme preparation and 50 µg of bovine serum albumin per 50 µl of reaction mixture. The opensquares represent the cleavage on the upperstrand, and the opencircles are the reaction on the lowerstrand. The reaction product was denatured and separated on a 20% urea-polyacrylamide DNA sequencing gel. Upon autoradiography, bands were cut out and measured for radioactivity. Values of the corresponding blanks were subtracted from the test values. Each point represents the average of two independent tests.



Characterization of 5-Methylcytosine-DNA Glycosylase

Fig. 3shows that 5-methylcytosine-DNA glycosylase has roughly the same affinity for hemimethylated oligonucleotide A and the non-methylated oligonucleotide (identical results were obtained with oligonucleotide containing a G/T mismatch; data not shown). The specific enzymatic activity recovered from bands a, b, and c are shown in Fig. 3, panelB. The enzyme activity was only found in the protein-DNA complex ``a'' for both the methylated and non-methylated oligonucleotide. UV cross-linking of the protein(s) in band ``a'' with oligonucleotide D (Table 1) shows the presence of two peptides of a molecular mass of 30 and 55 kDa, respectively (Fig. 3C). To ascertain which of the two peptides had the 5-methylcytosine-DNA glycosylase activity, proteins from post-Mono-S fractions were separated on a 10% SDS-polyacrylamide gel, and bands were extracted and tested as described under ``Materials and Methods.'' The peptide with a molecular mass of 52.5 kDa shown in Fig. 2, lane5, was the only one having the major activity of 5-methylcytosine-DNA glycosylase and the mismatch-specific thymine-DNA glycosylase (see also Fig. 1, middlepanel). Occasionally, we also found a smaller double band of an approximate molecular mass of 50 kDa having also both activities (Fig. 2, lane4). As we have previously shown, the combined action of 5-methylcytosine-DNA glycosylase and AP-endonuclease (the combined action of the two enzymes was called 5-metCpG-endonuclease) present in crude nuclear extracts from chicken embryos is not sequence specific but only metCpG specific(8) . The results of Fig. 4show that 5-methylcytosine-DNA glycosylase does not react with single-stranded oligonucleotide A methylated at a single CpG (lane3). Furthermore, the bifilarly methylated oligonucleotide B is a very poor substrate for the enzyme when compared with hemimethylated DNA (Fig. 4, compare lanes1 and 4). A comparison of the kinetics of cleavage of hemimethylated oligonucleotide A with the same bifilarly methylated oligonucleotide B is shown in Fig. 5(compare the kinetics of the closedcirclesversus the opencircles and squares) where there is up to a 6-fold difference in the rate of specific reaction between the two substrates tested with the same batch of purified enzyme. The presence of a G/T mismatch two base pairs away from the 5-methylcytosine (Table 1, oligonucleotide f) inhibits to some extent the 5-methylcytosine-DNA glycosylase, and reciprocally the presence of a 5 met-Cyt close to the G/T mismatch slightly inhibits the G/T mismatch glycosylase (see Fig. 1, compare lane2 of upper and lowerpanels). The maximal activity of 5-methylcytosine-DNA glycosylase is obtained between pH 6.5 and 7.5, and the P(i) of 5-methylcytosine-DNA glycosylase is 5.5-7.5 (data not shown). The K(m) for the labeled substrate A (Table 1) was measured with a Mono-S purified enzyme. The reaction product was analyzed by the standard gel shift assay, as described under ``Materials and Methods.'' Fig. 6shows the Lineweaver-Burk plot of the results. With substrate A, we have a K(m) of 8 times 10M with a V(max) of 4 times 10M/h/µg of protein. The exceedingly low turnover number of the enzyme that can be calculated from our data (2/h) could possibly be explained by the big loss of catalytic activity that we observed during the extraction, denaturation, and renaturation of the enzyme from preparative gels.


Figure 3: 5-Methylcytosine-DNA glycosylase binds to hemimethylated and non-methylated DNA. PanelA, gel mobility shift assay on 5% native polyacrylamide gel of post-affinity chromatography purified 5-methylcytosine-DNA glycosylase. Lane1 is the reaction with the hemimethylated oligonucleotide A, and lane2 is the reaction with the same non-methylated oligonucleotide. a and b are the protein-DNA complexes, and c is the free unbound DNA. PanelB, 5-methylcytosine-DNA glycosylase activity recovered from the complexes a and b and free DNA (c) from hemimethylated DNA (1 on figure) and non-methylated DNA (2 on figure). S are the size standards. The product of reaction was separated on a 20% urea-polyacrylamide denaturation gel. PanelC, UV cross-linking of protein-DNA complexes formed in band a from hemimethylated oligonucleotide D (1a) and from the same non-methylated oligonucleotide (2a). 1c is a control of hemimethylated DNA incubated without protein. The reaction product was analyzed on a 10% SDS-polyacrylamide gel. bp, base pairs.




Figure 2: Polypeptide profiles (on 10% SDS-polyacrylamide gel) at different stages of purification of 5-methylcytosine-DNA glycosylase. 3 µg of protein (for lanes1-4) were separated and silver stained. Lane1 is the crude nuclei extract, lane2 is the fraction post-heparin-Sepharose, lane3 is the post-DEAE-CM-Sepharose fraction, lane4 is the post-Mono-S chromatography fraction, and lane5 is the 5-methylcytosine-DNA glycosylase eluted from a preparative SDS-polyacrylamide gel. The molecular mass markers (Bio-Rad) are phosphorylase b, 97 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; and carbonic anhydrase, 30 kDa. On the rightside, we show the activity of 5-methylcytosine-DNA glycosylase (mC) and mismatch-specific thymine-DNA glycosylase (G/T) recovered from single peptide bands. Enzyme assays were carried out as outlined under ``Materials and Methods.'' The arrows represent the band(s) that were excised, extracted, and tested for enzymatic activities




Figure 4: 5-Methylcytosine-DNA glycosylase reacts preferentially with hemimethylated DNA (lane 1 with corresponding control in lane 2) and very poorly with single-stranded methylated DNA (lane 3) or with bifilarly methylated DNA (lane 4). Lane5 is the control of lane4, incubated without protein. For hemimethylated DNA (oligonucleotide A) and bifilarly methylated DNA (oligonucleotide B), both strands were labeled at the 5`-ends with the kinase reaction. The expected cleavage product for the upper and lower strands are 21 and 28 bases, respectively. For this experiment, 2 µg of a post DEAE-CM-Sepharose fraction were incubated in the presence of bovine serum albumin. The cleavage product was analyzed on a 20% urea-polyacrylamide DNA sequencing gel. In laneS, the size standards in base pairs are shown. bp, base pairs.




Figure 6: Determination of the Kand V(max) for the hemimethylated DNA substrate A incubated with purified 5-methylcytosine-DNA glycosylase (fraction post-CM-Sepharose). Each 50 µl of incubation mixture (in duplicates) received 2 µg of protein and increasing concentrations of end-labeled oligonucleotide A. After 1 h of incubation at 37 °C, samples were analyzed on a 20% urea-polyacrylamide DNA sequencing gel. Upon autoradiography, bands were cut out and counted for radioactivity. Corresponding controls, with the DNA substrate only, were also incubated, and an area corresponding to the protein-DNA complex of the test was cut out, counted, and subtracted from the test values. Results are expressed as a Lineweaver-Burk plot.



In vivo modification/demodification of DNA has been shown to start at discrete positions in the promoter region and to proceed bidirectionally(3) , suggesting, in the case of active demethylation, that the enzymes may be processive. To test this hypothesis, a 69-base pair-long oligonucleotide containing 5 mCpGs was synthesized (Table 1, oligonucleotide H), and the end-labeled DNA was incubated for various times with purified 5-methylcytosine-DNA glycosylase (post-CM-Sepharose chromatography). The results of Fig. 7show the reaction product obtained after 15, 30, 45, and 60 min of incubation. Even for the shortest incubation time, there is no sign of processivity of the enzyme for the five different mCpGs. However, some sites seem to be a better substrate for the enzyme than others (Fig. 7, compare mCpGs at positions 21 and 28). The differences observed were not due to the extent of DNA methylation (data not shown).


Figure 7: 5-Methylcytosine-DNA glycosylase is distributive. Oligonucleotide H (Table 1) was end-labeled and incubated with 2 µg of post-CM-Sepharose fraction. Incubation was carried out at 37 °C of 15, 30, and 45 min, respectively (lanes1-3). A control incubated without protein for 45 min is shown in lane4. S values are the size standards. Samples were processed and analyzed on a 20% urea-polyacrylamide DNA sequencing gel, as indicated under ``Materials and Methods.''



Characterization of the Reaction Product

The identity of the DNA cleavage product produced by the affinity-purified 5-methylcytosine-DNA glycosylase was tested on TLC plates outlined under ``Materials and Methods.'' Fig. 8shows that the most abundant cleavage product is ^3H-labeled 5-methylcytosine. Th is result is strong, supporting evidence for 5-methylcytosine-DNA glycosylase activity. Moreover, as indicated by the presence of low levels of [^3H]thymine, the enzyme preparation had very little 5-methylcytosine deaminase activity. Following the removal of the base from the sugar by the glycosylase, the apyrimidic site is cleaved by an AP-endonuclease, leaving a single strand break in the DNA. In vitro, the apyrimidic sites can be cleaved by alkaline hydrolysis. Fig. 1(upperpanel, lanes1 and 2) shows that the 5`-end-labeled oligonucleotide A, which had been incubated with 5-methylcytosine-DNA glycosylase when hydrolyzed with 0.1 M NaOH, gave one base shorter DNA fragment. This result suggests that the AP-endonuclease cleaves 3` from the apyrimidic site. The same observation was made for the cleavage site of the G/T mismatch (Fig. 1, upperpanel, lanes5 and 6), where the cleavage was T specific and did not occur on the opposite G residue (Fig. 1, lowerpanel, lanes5 and 6). The nature of the adjacent base 3` from the apyrimidic site was determined as outlined under ``Materials and Methods.'' Fig. 9shows that the base 3` from the apyrimidic site is a guanosine residue. This means that only the 5-methylcytosine is removed from the methylated CpG site.


Figure 8: Reaction product of 5-methylcytosine-DNA glycosylase analyzed by TLC. 2 µg of enzyme preparation eluted from an affinity matrix were incubated with ^3H-labeled (5-methylcytosine) hemimethylated DNA for 1 h at 37 °C, and the reaction product was separated as indicated under ``Materials and Methods.'' Each bar diagram represents the average of three separate analyses. bp, base pairs.




Figure 9: Nature of the base present 3` from the apyrimidic deoxyribose (at the CpG site). Hemimethylated DNA was incubated under the same conditions as in Fig. 8, and the resulting cleavage product was analyzed by TLC as described under ``Materials and Methods.'' Each bar represents the average of four different determinations.




DISCUSSION

If we compare the present results with our previously published data(8) , there is an apparent contradiction. While using crude nuclear extracts from chicken embryos, we could show a specific cleavage at the mCpGs, but we could not demonstrate the presence of a specific 5-methylcytosine-DNA glycosylase. Now, upon purification of the mCpG-cleaving activity, we can clearly see the presence of a specific 5-methylcytosine-DNA glycosylase. The reason for this discrepancy, between our previous and actual results, is that the incubation of the labeled substrate with crude nuclear extracts had to be carried out for several hours at high concentrations of proteins, where the released 5-methylcytosine was immediately converted into thymine by a 5-methylcytosine deaminase. As far as we can judge, there is very little to no 5-methylcytosine deaminase activity in our highly purified 5-methylcytosine-DNA glycosylase preparations. The presence of very active 5-methylcytosine deaminase in vertebrate cells has already been reported recently by Vairapandi and Duker (9) who found in HeLa cells a very rapid conversion of 5-methylcytosine into thymine. We also reported (8) that the reaction product after lengthy incubations with crude nuclei extracts was not sensitive to alkaline hydrolysis, presumably because the apyrimidic sugar had been removed during the incubation. In contrast, we now show that the reaction product from an incubation mixture containing the highly purified 5-methylcytosine-DNA glycosylase is 5-methylcytosine, and the DNA is sensitive to the alkaline hydrolysis, indicating that the apyrimidic deoxyribose is still present in the DNA.

However, there is still a major difference between our results and those reported by Vairapandi and Duker(9) . The 5-methylcytosine-glycosylase purified from chicken embryos cleaved preferentially hemimethylated DNA, whereas Vairapandi and Duker (9) found just the opposite. In their case, bifilarly methylated DNA is clearly a better substrate than hemimethylated DNA. The reasons for this difference are, at present, not known. Like the DNA-methyltransferase, the purified 5-methylcytosine-DNA glycosylase reacts much faster with hemimethylated DNA than with the non-methylated or fully methylated DNA, respectively, suggesting that the two enzymes may compete for the same substrate during replication. In this case, the mole ratio of the two enzymes combined with their respective binding affinities to DNA may decide which of the two enzymes will react with the newly replicated DNA, i.e. methylation or demethylation. It is interesting to note that prior to the genome-wide demethylation occurring in some differentiating systems, there is an abrupt drop in the methyltransferase activity(7, 17) . In this case, it is possible that the drop in DNA methyltransferase activity prevents the maintenance methylation of the newly replicated DNA, which then becomes the substrate for the 5-methylcytosine-DNA glycosylase. This way it may be possible to demethylate completely a specific site on DNA after just one round of replication. The site specificity of the reaction may be directed by specific transcription factors (18) and/or cis-acting elements(4, 5) .

Our most surprising result is the copurification of 5-methylcytosine-DNA glycosylase with the mismatch-specific thymine-DNA glycosylase. At present, it is not possible to decide whether a single peptide has the two enzymatic activities or whether two very close related peptides with different enzymatic activities copurify throughout the purification scheme. As we already mentioned under ``Results,'' we also observed on SDS-polyacrylamide gels that some preparations of the highly purified 5-methylcytosine-DNA glycosylase had minor bands migrating with an apparent molecular mass of 50 kDa. These minor bands had also the same enzyme activity ratio of 5-methylcytosine-DNA glycosylase and mismatch-specific thymine-DNA glycosylase and could represent either a degradation product of the 52.5-kDa peptide or covalent modified forms of the larger peptide. Clearly, we will have to wait until we have microsequenced these peptides and cloned the cDNAs to decide whether we are dealing with one peptide having the two activities or whether we have two separate enzymes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 61-6976688; Fax: 4161-7214091.

(^1)
J.-P. Jost, unpublished results.

(^2)
The abbreviations used are: AP, apyrimidine; FPLC, fast protein liquid chromatography; DTT, dithiothreitol.


ACKNOWLEDGEMENTS

We thank Drs. Alain Bruhat, Jan Hofsteenge, and Frédéric Schmitt for constructive discussions and Y.-C. Jost for typing the manuscript. We are grateful to D. P. Schofield for editing of the manuscript.


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