We Have Purified 3-Methyladenine DNA Glycosylase I from Escherichia Coli to Apparent Physical Homogeneity

Voue1Volume 15 Numberubr7187 1987 NucleicNcecAisRsacAcids Research

Purification and characterization of 3-methyladenine DNA glycosylase I from Escherichia coli

Svein Bjelland and Erling Seeberg

Norwegian Defence Research Establishment, Division for Environmental Toxicology, PO Box 25, N-2007 Kjeller, Norway

Received February 16, 1987; Accepted March 2, 1987

ABSTRACT We have purified 3-methyladenine DNA glycosylase I from Escherichia coli to apparent physical homogeneity. The enzyme preparation produced a single band of Mr 22,500 upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis in good agreement with the molecular weight deduced from the nucleotide sequence of the tag gene (Steinum, A.-L. and Seeberg, E. (1986) Nucl. Acids Res. 14, 3763- 3772). HPLC confirmed that the only detectable alkylation product released from (3H)dimethyl sulphate treated DNA was 3-methyladenine. The DNA glycosylase activity showed a broad pH optimum between 6 and 8.5, and no activity below pH 5 and above pH 10. MgSO4, CaCl2 and MnC12 stimulated enzyme activity, whereas ZnSO4 and FeC13 inhibited the enzyme at 2 mM concentration. The enzyme was stimulated by caffeine, adenine and 3-methylguanine, and inhibited by p- hydroxymercuribenzoate, N-ethylmaleimide and 3-methyladenine. The enzyme showed no detectable endonuclease activity on native, depurinated or alkylated plasmid DNA. However, apurinic sites were introduced in alkylated DNA as Judged from the strand breaks formed by mixtures of the tag enzyme and the bacteriophage T4 denV enzyme which has apurinic/apyrimidinic endonuclease activity. It was calculated that wild-type E. coli contains approximately 200 molecules per cell of3-methyladenine DNA glycosylase I.

INTRODUCTION One ofthe major lesions formed in DNA exposed to simple alkylating agents such as methylmethane sulphonate and N-methyl-N-nitrosourea is 3-methyladenine (1). DNA glycosylases which release 3-methyladenine from alkylated DNA were first discovered in bacteria (2-4), but have since been detected in many different organisms, including human cells (for review see ref. 5). Isolation and characterization of Escherichia coli mutants lacking 3-methyladenine DNA glycosylase activity have shown that 3-methyladenine represents the major cytotoxic lesion introduced by alkylation (6,7). This is further indicated by the fact that E. coli possesses two distinct DNA glycosylases which both release 3-methyladenine from alkylated DNA (8,9). One is constitutively expressed and encoded by the tag gene (TagI) whereas the other is inducible and encoded by alkA (Tagil, ref. 9). The alkA gene function is part ofthe adaptive response to alkylating agents and is positively regulated by ada (9-12).

Both the tag and alkA genes have been cloned and sequenced (10,12-14). The amino acid sequences derived from the nucleotide sequences give proteins of Mr 21,104 and 31,402, respectively (13,14). This corresponds to the molecular weights determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis of radiolabelled polypeptides expressed from the cloned genes (12,15). Further, the alkA coded glycosylase has been purified to apparent homogeneity giving a single protein band of 31,000 Da on gels (12). A strain carrying a plasmid which overproduces the alkA enzyme was used as enzyme source. Purification of the tag enzyme has also been reported but homogeneous preparations were not obtained because of enzyme lability and because of limited amounts of enzyme present in normal wild-type cells (4,16). In the present work we describe a new procedure for purification of the tag enzyme. The enzyme was stabilized by high concentrations of glycerol in the elution buffers and we used as an enzyme source a strain carrying the tag gene on a multicopy plasmid which overproduces the enzyme about 100-fold. The purification gives apparently homogeneous enzyme in 30% yield.

MATERIALS AND METHODS Bacterial strains and plasmids Strain MS23 (alkAl, ref. 17) transformed by pBK202 (tag+) was used as enzyme source (14,15). The alkAl genotype ofthe host was chosen to avoid any interference with TagIl activity during purification. ColEl plasmid DNA was isolated from SK2001 (18). Both strains are derivatives ofE. coli K12. Assay of3-methyladenine DNA itlycosylase activity Activity of 3-methyladenine DNA glycosylase was measured according to Riazuddin and Lindahl (4) with minor modifications. Substrate was (3H)dimethyl sulphate (1.8 Ci/mmol, New England Nuclear, U.S.A.) treated calf thymus DNA (1.2 x 104 cpm/pg). Standard reaction mixtures contained 0.3 pg DNA (5 P1 in 0.01 M Tris-HCl, pH 8/1 mM EDTA), 70 mM Mops, pH 7.5/1 mM EDTA/1 mM dithiothreitolV5% (v/v) glycerol (reaction buffer) and enzyme in a total volume of 50 p1. The mixtures were incubated at 37°C for 30 min followed by addition of 50 pl ofwater, 20 p1 1.5 M sodium acetate/0.1% (w/v) calf thymus DNA, 3 pl of 20 mg/ml bovine serum albumin and 300 pl 100% ethanol. The DNA was precipitated by chilling for at least 20 min at -70°C before centrifugation at 10,000 x g for 10- 15 mmn. Released 3-methyladenine present in the supernatant was determined by liquid scintillation counting. For assay of dilute enzyme solutions 1 mg/ml of bovine serum albumin was added to the reaction buffer. To determine the activity as a function ofpH, an universal buffer (19) ofdifferent pH values containing 1 mM EDTA/1 mM dithiothreitol/5% (v/v) glycerol (buffer U) was used as reaction buffer.

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One unit of 3-methyladenine DNA glycosylase is defined here as the amount of enzyme that catalyses the release of 1 pmol of 3-methyladenine during the standard reaction time of30 min. Assay ofAP endonuclease activity AP endonuclease activity was assayed by detecting break formation in covalently closed circular DNA in which AP sites had been introduced by heating at low pH (20). AP sites were introduced by adding ColEl plasmid DNA (8 pg in 10 p1 0.01 M Tris-HCl, pH 8/1 mM EDTA) to 40 pl of 0.01 M sodium citrate buffer, pH 5 containing 0.1 M NaCl, followed by heating at 700C for 20 min. The reaction was stopped by the addition of 50 pl buffer U, pH 6.7. The same amount of plasmid diluted 1:10 in buffer U was used as control. The plasmid solutions above (8 pI) were incubated with 2 pg ofpurified enzyme in buffer U in a total volume of200 p1 at either pH 6 or pH 8 at 370C for 30 min followed by incubation with 10 pg (in 10ll water) of proteinase K (Boehringer Mannheim GmbH, F.R.D.) for 10 min. As a positive control apurinic DNA was also incubated with 2 pg ofT4 endonuclease V. The DNA was concentrated by ethanol precipitation and analysed by agarose gel electrophoresis. Enzyme reaction with alkylated plasmid DNA ColEl plasmid DNA (1.6 pg) was alkylated by adding 4 p1l of methylmethane sulphonate (Aldrich, F.R.D.) to the DNA in 600 pl of 10 mM potassium phosphate buffer, pH 7.5 and exposed for 8 min at 370C. The reaction was stopped by chilling on ice and precipitating the DNA with ethanol. Another sample without methylmethane sulphonate was incubated as control. Both the alkylated and the control DNA were dissolved in 400 pl ofbuffer U, pH 6, and split in four parts. One of each was incubated at 370C for 30 min with 0.4 pg of Tag alone, 0.4 pg Tag and 0.1 pg ofT4 endonuclease V, with 0.1 pg ofT4 endonuclease V alone and with 2 p1 of 0.1 M Tris-HCl, pH 8.5 containing 1 mM EDTA/10 mM 2-mercaptoethanoll20% (v/v) glycerol as control. The enzymes were degraded by incubation with 10 pg (in 10 pl water) of proteinase K for 30 min. The DNA was concentrated by ethanol precipitation and analysed by agarose gel electrophoresis. HPLC of alkylated bases For HPLC of excision products the alkylated calf thymus DNA (15pg) was further purified on Nensorb 20 (using conditions as recommended by the manufacturer, Du Pont Company, U.S.A.) to remove any alkylated bases released spontaneously during storage. Repurified alkylated DNA (7.5 pg) was incubated with 16 ng of Tag, or with buffer alone as control, in a total volume of 300 p1 of reaction buffer at 370C for 15 min, followed by DNA precipitation with ethanol. The supernatants were concentrated by evaporation, mixed with alkylated bases as markers and subjected to HPLC. The precipitate of the DNA incubated with

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buffer alone was dissolved in 100 p1l of 0.1 M HCl and incubated at 37°C for 24 h to release alkylated bases by hydrolysis. The pH was adjusted to 7.1 with NaOH, and the DNA precipitated with ethanol. The supernatant was concentrated by evaporation and subjected to HPLC. Another experiment was designed to optimize conditions for the possible release of3-methylguanine. Repurified DNA (14 pg) was first incubated with 64 ng ofTag in 180 p1l ofreaction buffer for 15 min for complete removal of 3-methyladenine from the alkylated DNA. A second portion of enzyme (64 ng) was then added in a total volume of 300 p1l and incubation continued for another 15 min. As controls were included one sample to which no enzyme was added, and another including enzyme only for the first incubation. HPLC of alkylated bases was performed on a reverse phase column (Spheri-5 RP-18, 22 x 4.2 mm) from Brownlee Labs Inc., U.S.A. The column was eluted with a linear gradient of97-90% 0.1 M triethylammoniumacetate buffer in methanol (v/v) over a period of 10 min, followed by 90-75% (v/v) triethylammoniumacetate over a period of 5 win. The flow rate was 1.5 ml/min. The pH of the triethylammoniumacetate buffer was either 5.4 or 7.5. At pH 5.4 good separation is obtained between 3- methyladenine and 7-methylguanine (as presented in figure) while pH 7.5 gives good separation of 3-methyladenine and 3-methylguanine (used for the second experiment). The retention times for 3-methyladenine, 3-methylguanine, 6- methylguanine and 7-methylguanine at pH 5.4 was 5.3, 5.7, 14.1 and 8.2 min, and at pH 7.5 7.4, 5.7, 13.7 and 8.0 min, respectively. Fractions were collected and the radioactivity measured in a liquid scintillation counter. Purification of plasmid DNA Covalently closed circular ColEl DNA was purified by an upscaling of the alkaline extraction method ofBirnboim and Doly (21). E. coli SK2001 was grown at 370C in K medium (175 ml) containing 50 pg/ml of thymine until a density of 2 x 108 cells per ml. Chloramphenicol (100 pg/ml) was added and the incubation continued for 20 h. The cells were washed in 0.04 M Tris-HCl, pH 8, concentrated by centrifugation (7,500 x g 5-10 min) and resuspended in 4 ml ice-cold 2 mg/ml lysozyme/10 mM EDTA/25 mM Tris-HCl, pH 8/50 mM glucose. After incubation on ice for 20 min, 8 ml of 1% (w/v) sodium dodecyl sulphate in 0.2 M NaOH was added and the lysate left on ice for an additional 5 min. The solution was then neutralised by adding 6 ml of 3 M sodium acetate, pH 4.8, followed by centrifugation (20,000 x g 15 min), to remove most cell debris and chromosomal DNA. The plasmid DNA was precipitated twice with ethanol. The pellet was dissolved in 3.9 ml of 0.01 M Tris-HCl buffer, pH 8/1 mM EDTA and the material was banded twice in CsCV/ethidium bromide using a vertical Sorwall TV-865 rotor. The ethidium bromide was removed by several extractions with isopropanol/1 M Tris-HCl, pH 8

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(20:1 (v/v)) followed by extensive dialysis against 0.01 M Tris-HCl, pH 8/1 mM EDTA. Agarose gel electrophoresis Agarose gel electrophoresis was carried out on a horizontal 0.7% (w/v) slab gel (0.7 x 7 x 10 cm) with wells of 1 x 2 mm. The running buffer was 0.089 M Tris- borate, pH 8/2 mM EDTA. Following electrophoresis the gels were stained in 5 pg/ml of ethidium bromide. The agarose (Ultra Pure, No. 5510UA) was a product ofBethesda Research Laboratories, Inc., U.S.A. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis Electrophoresis was run according to Laemmli (22) using a vertical Protean II slab cell from Bio-Rad Laboratories, U.S.A. The experiment was carried out as recommended by the manufacturer with a separation gel of 15% (w/v) polyacrylamide (0.15 x 16 x 12 cm). The samples were heated at 950C for 3 min. Electrophoresis was performed at 40 V for 40 min followed by 70 V for 2 h and finally 150 V for 4 h. The gel was fixed with 10% (w/v) trichloroacetic acid/10% (v/v) acetic acid/30% (v/v) methanol, followed by staining in 0.1% (w/v) Coomassie brilliant blue R/20% (w/v) trichloroacetic acid for 1 h. Destaining was performed with 20% (v/v) methanolV10% (v/v) acetic acid. Coomassie brilliant blue R was a product (No. B-0630) of Sigma Chemical Company, U.S.A. The protein markers were the Low Molecular Weight Protein Standards of Bio-Rad: 50 P1 of a 1:280 dilution was applied to each track. Other methods and general information All centrifugations and step 2 were carried out at 40C. Steps 3 and 4 were performed under refrigeration below 100C. The FPLC system with the Mono S and Mono Q columns are products of Pharmacia AB, Sweden. All pH values indicated are those determined at room temperature. Protein concentration was determined by the method of Bradford (23) using the Bio-Rad Protein Assay Kit with bovine serum albumin as standard. All chemicals were analytical grade.

RESULTS Purification ofTagI Specific activity and recovery of the enzyme at various steps of the purification are indicated in Table 1. Preparation ofcell extract. Cell extracts were made by a combination of sucrose plasmolysis and lysozyme treatment as described which yields a protein extract essentially free ofcontaminating chromosomal DNA (24). Cells were grown at 37°C in K medium (1.9 1) containing 25 pg/ml ampicillin and harvested by centrifugation (10,000 x g 5-10 min) at a density of 5 x 108 cells per ml. The cells

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Table 1. Purification of3-methyladenine DNA glycosylase I from E. coli.

|Fraction Volume Protein Specific Purification Recovery Fraction (ml) (mg) acunity/i (fold) (%)

I. Protein extract 51 165 700 1 100 HI. Gelfiltration on Ultrogel AcA 21.5 5.6 10,000 14 49 54 m.Cation exchange chromato- 1.5 0.35 140,000 200 43 graphy on Mono S HR 5/5 IV.Anion exchange chromato- 1 0.15 220,000 315 29 graphy on Mono Q HR 5/5 were washed once in 0.04 M Tris-HCl, pH 8, collected by centrifugation (7,500 x g 5 min) and resuspended at ice temperature in 12 ml of84% (w/v) sucrose (dissolved in 40 mM Tris-HCl, pH 8/10 mM EGTA). The sucrose suspension was left on ice for 10 min whereafter 48 ml of 50 mM Mops, pH 7.5/100 mM KCII1 mM EDTA/1 mM dithiothreitol containing 125 pg/ml lysozyme was added and the mixture further incubated on ice for 45 min. Cell debris and DNA were subsequently removed by centrifugation for 15 min at 27,000 x g and the extract stored frozen until use (Fraction I). Gelfiltration on Ultrogel AcA 54. A column ofUltrogel AcA 54 (2.5 x 43 cm, LKB Produkter AB, Sweden) was equilibrated with 0.1 M Tris-HCl, pH 8.5/1 mM EDTA/10 mM 2-mercaptoethanol/400 mM KCI/20% (v/v) glycerol. Fraction I was first subjected to ammonium sulphate fractionation to achieve a crude enrichment of the enzyme and to concentrate the sample (4). Solid ammonium sulphate was slowly added under gentle stirring on an ice bath to a final concentration of 45% saturation. After additional stirring for 30 min, the precipitate was removed by centrifugation at 27,000 x g for 15 min. Solid ammonium sulphate was added to the supernatant as above to a final concentration of 70% saturation, followed by stirring and centrifugation. The precipitate was resuspended in 5 ml of equilibration buffer and applied to the column. The column was eluted at a flow rate of0.35 ml/min. Active fractions were pooled and desalted using Sephadex G-25 M (Column PD-10, Pharmacia) equilibrated with 50 mM Mes buffer, pH 6/1 mM

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40- 020.4E

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activity-(l) prtincnenrton()

Fpig. 1.CatioFPLCexhnecrmtgahIFL)onMnonMono QHR 5/5 ofuimehratdeninlR55DN MesyufaercinII fro EMa

coli.Conition werdfescrlibedtionas thfertextoandfrac0mlonsiz winas0.5amlienzyme0 frativityCAtion(0);exchangeprotein concchromatographyentation (o). (FPLC)oumon MonoD1S HRqulbae5/5. Fractionwt rm.was applied to FPLC Mono s HR 5/5 equilibrated with 0.mM Mies bufe, pH 5/1 mM EDTA/10Ti-C,p8./mMEDTA/10mM 2-mercaptoethanol/20%mM 2-mercaptoethanoV/20%(v/v) glycerol.(v/v)Theglycerol.Theenyewsprfd14columnwas eluted(0. ml/min) with ml5 ofequilibration buffer followed by 10 ml ofa linear gradient of0- fnotldb tenzgelfltactionvith50%cio rEcoey(Faton. 200 mM NaClin the same buffer. The enzyme eluted at about 0.14 M NaCl. Active fractions were pooled and desalted using Column PD-10 equilibrated with 0.1 M Tris-HCI, pH 8.5/1 mM EDTA/10 mM 2-mercaptoethanoll20% (v/v) glycerol. The cation exchange chromatography gave 14-fold purification with only a minor loss in total enzyme activityIs).(Fraction Anion exchange chromatography (FPLC) on Mono Q HR 5/5. Fraction III was applied to FPLC Mono Q HR 5/5 equilibrated with 0.1 M Tris-HCI, pH 8.5/1 mM EDTA/10 mM 2-mercaptoethanol/20% (v/v) glycerol. The column was eluted (0.2 ml/min) with 5 ml ofequilibration buffer followed by 10 ml of a linear gradient of 0-200 mM KCi in the same buffer. The enzyme eluted at about 0.14 M KCI and the activity profi'le corresponded to the protein profile, indicating homogeneous protein (Fig. 1). The final preparation (Fraction IV) was purified 300-fold relative to the cell extract with 30% recovery. This corresponds to an increase in specific activity of 30,000 relative to the activity found in extracts from wild-type E. coli. General properties Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the various fractions obtained at different steps of the purification showed a distinct band of

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Fig. 2. Sodium decvl sulphate/polvacrvlamide gel electrophoresis of Drotins in the fractions obtained during purification of3-methyladenine DNA g-ycosylase I of E. coli. The separation gel was 15% (w/v) polyacrylamide, and the gel was stained with Coomassie brilliant blue. Each track (from the left) represents: 1 and 6, molecular weight markers (from the top) phosphorylase B (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400); 2, Fraction I (crude extract, 16 pg); 3, Fraction II (Ultrogel, 6.2 pig); 4, Fraction III (Mono S, 5.6 pg); 5, Fraction IV (Mono Q, 3.4 plg). The bands at the top common to all the lanes (best visualised in control track 6) are artifacts ofthe electrophoresis procedure.

TagI even in the crude extract from the overproducing strain (Fig. 2). A high degree of purity was obtained in Fraction Im (after Mono S), whereas Fraction IV appeared homogeneous, as could be expected from the overlapping protein and activity profiles of the eluate from the last column (Fig. 1). TagI migrated as a protein of Mr 22,500 in the sodium dodecyl sulphate/polyacrylamide gel electrophoresis system used, which is in reasonable agreement with the exact molecular weight of 21,104 deduced from the nucleotide sequence of the tag gene (14). The purified enzyme had a broad pH optimum between pH 6 and 8.5, and no activity below pH 5 and above pH 10 (Fig. 3). The divalent metal salts MgSO4, CaCl2 and MnCI2 stimulated Tag activity to approximately the same extent at a 2mM concentration, whereas the divalent ZnSO4 and the trivalent FeC13 were inhibitory at the same concentration (Table 2). It is well established that TagI is product inhibited by 3-methyladenine (4,16). However, some other purine analogs,

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&L200 S

z 100- I

3 4 5 6 7 8 9 10 pH

Fig. 3. Effect of pH on 3-methyladenine DNA glycosylase I activity. Incubation was performed with 1.6 ng ofpurified enzyme at 37°C for 10 min. i.e. caffeine, adenine and 3-methylguanine, appear to have a stimulatory effect (Table 2). TagI contains as many as 8 cysteine residues, and some of these may be involved in maintaining protein structure by forming disulphide bridges (14). However, the enzyme also contains essential sulfhydryl groups as sulfhydryl- blocking agents like p-hydroxymercuribenzoate and N-ethylmaleimide have inhibitory effect (ref. 4,16, Table 2). The purified enzyme was stable for more than six months when stored frozen at -20°C diluted in reaction buffer containing 1 mg/ml of bovine serum albumin. Repeated freezing and thawing during this period did not effect any apparent loss in enzyme activity. For long term storage the enzyme was kept at -70°C in the final buffer containing 20% (v/v) glycerol. Reverse phase HPLC ofenzyme-catalysed release ofalkylated bases The assay used for the enzyme purification simply measures overall release of ethanol-soluble methylated bases from dimethyl sulphate treated DNA. Experi- ments with partially purified enzyme have shown that 3-methyladenine is the only detectable alkylation product in the ethanol-soluble fraction (2,4,16). We have used reverse phase HPLC to analyse the composition of the excised alkylated bases in the supernatant after ethanol precipitation of dimethyl sulphate treated DNA incubated with the purified enzyme. In the first set of experiments the eluting conditions were chosen to obtain optimal separation of 3-methyladenine and 7-methylguanine. The results confirmed that only 3-methyladenine and no 7-methylguanine were excised by the

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Table 2. Effect of metal ions, sulfhydryl-blocking agents and purine base derivatives on 3-methyladenine DNA glycosylase I of E. coli. Incubation was performed with 0.4-0.6 ng ofTag.

Concentration Addition (MM) Enzymeactivity

no 100 MgSO4 2 155 CaCl2 2 150 MnCl2 2 130 FeCl3 2 40 ZnSO4 0.1 100 2 15 100 0 p-hydroxymercuribenzoate 1 7 N-ethylmaleimide 1 90 caffeine 5 145 20 145 adenine 5 155 3-methyladenine 5 50 3-methylguanine 5 130

enzyme (Fig. 4). In the other set of experiments the system was optimized to give much better separation of3-methyladenine and 3-methylguanine (data not shown). However, also in these experiments the only detectable excision product was 3- methyladenine; no significant release of 3-methylguanine was observed, in agreement with previous investigations (4,16). The fraction of7-methylguanine, 3- methyladenine and 3-methylguanine in the dimethyl sulphate treated DNA was analysed by acid depurination and HPLC to be 75%, 14% and 1.3%, respectively, of the total alkylated residues. As little as 10-15% excision of the total 3-methylguanine present in the substrate would have been detected in our experiments. Search for AP endonuclease activity in the enzyme preparation Some DNA glycosylases have associated AP endonuclease activity, e.g. the pyrimidine dimer DNA glycosylase (endonuclease V, denV enzyme) of bacteriophage T4 (25) and thymine glycol DNA glycosylase (endonuclease M) ofE. coli (26). Possible AP endonuclease activity associated with TagI was assayed by

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E 0.

400-

200-

5 10 15 ELUTION TIME (min) Fig. 4. Reve phase HPLC of methvlated bases released by 3-methyladenine DNA 1 cos lase I f ) mehy Sulphate treated calf us DNA. DNA (7.5 pg) was incubated with 16 ng of enzyme at 37°C for 15 min. The DNA was precipitated with ethanol and the supernatant was analysed by HPLC. Radio- activity in each fraction (0.3 ml) was measured in a liquid scintillation counter (0). Incubation was also performed without enzyme added as control (o). incubating the enzyme with covalently closed ColE1 DNA depurinated by brief acid treatment. Strand break formation in the DNA was analysed by agarose gel electrophoresis (Fig. 5). Incubation was carried out both at pH 6 and at pH 8 since it has been shown that the AP endonuclease activity of the denV enzyme had a sharp optimum at pH 6 (27). Our results showed that the TagI preparation did not change the superhelical form of the plasmid DNA, implying that TagI is devoid of any endonuclease activity on either native or depurinated DNA. Strand breaks were formed, however, specifically into depurinated DNA by the denV enzyme (Fig. 5). Introduction ofAP sites in alkylated DNA DNA glycosylase action results in the formation of an AP site. The introduction of AP sites into alkylated DNA by the purified TagI enzyme was demonstrated by mixed incubations with the denV enzyme. Superhelical ColEl DNA was treated with methylmethane sulphonate, incubated with the TagI and/or the denV enzyme, and analysed by agarose gel electrophoresis (Fig. 6). Some breakage of the superhelical DNA was introduced by the alkylating agent. Neither TagI, nor the

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Fig. 5. Agarose ael electroPhoresis of native or depurinated RFI DNA incubated with TagI or the denV enzyme of bacteriop age T4. The fast-mi rating form (top) is RE whereas the slow-migrating species represents DNA with nicks (RFII). Lanes 1-3 represent native DNA incubated with buffer alone, pH 6 (1), TagI, pH 6 (2) and denV, pH 6 (3) whereas lanes 4-8 represent DNA with AP sites incubated with denV, pH 6 (4), TagI, pH 6 (5), TagI, pH 8 (6), buffer alone, pH 6 (7) and buffer alone, pH 8 (8). Electrophoresis was performed at 70 V for 2 h using 0.13 pg DNA per well; anode at the top. denV enzyme, caused any further breakage, however, mixtures of the enzymes introduced strand breaks specifically into alkylated DNA.

DISCUSSION The 3-methyladenine DNA glycosylase I ofE. coli has been purified to apparent physical homogeneity from an overproducing strain carrying the tag gene on a multicopy plasmid. The glycosylase action of the purified enzyme was demonstrated both by the positive identification of3-methyladenine as the excision product and by the enzymatic detection ofAP sites formed into alkylated DNA. Reverse phase HPLC indicated that 3-methyladenine is the only alkylation product released by TagI from dimethyl sulphate treated DNA, in agreement with previous reports (4,16). It has recently been shown that high expression of TagI will releave the alkylation sensitive phenotype of alkA mutant cells (15). The product of the alkA gene, Tagil, can remove 3-methylguanine as well as 3- methyladenine from alkylated DNA and the alkylation sensitive phenotype of

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1 2 3 4 5 6 7 8

Fig. 6. Agarose gel electrophoresis of alkylated RFI DNA incubated with TagI and/or the denV enzyme. Conditions were as described in legend to Fig. 5 except that 0.08 pg DNA was applied to each well. Lanes 1-4 represent methylmethane sulphonate treated DNA incubated with buffer alone (1), TagI (2), denV (3) and TagI + denV (4), whereas lanes 5-8 represent native DNA incubated with enzymes in the same order as for lanes 1-4, respectively. Electrophoresis was performed at 30 V for 20 min, followed by 70 V for 2 h; anode at the top. alkA mutant cells has been ascribed to the lack of repair of the minor alkylation product 3-methylguanine. It is therefore somewhat surprising that high levels of TagI can substitute for the lack ofTagil. One possible explanation was that TagI at a low rate also would remove 3-methylguanine, but that this activity had escaped detection. We therefore looked carefully for such activity using a high amount of enzyme, but with negative results. Incubation was also carried out twice, to release all 3-methyladenine during the first incubation. Nevertheless, we could not reveal any release of 3-methylguanine during the second incubation. This demonstrates that the enzyme does not release 3-methylguanine under conditions where competition with 3-methyladenine-containing DNA should be minimized. The enzyme contained no detectable AP endonuclease activity (Fig. 5). This accords with the observation that amino acid sequences assumed to be associated with this activity in T4 endonuclease V (28) is not present in TagI (14). Nor is there any sequences of the type lys-trp-lys or lys-tyr-lys; tripeptides which cause

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phosphodiesterbond cleavage at AP sites in vitro (29,30). However, TagI has sequence homology to the T4 denV enzyme in a region thought to be associated with glycosylase activity (14), which may indicate similar active sites for the N- glycosylic bond cleavage in the two enzymes. The specific activity of 3-methyladenine DNA glycosylase I in extracts from wild- type E. coli was found to be approximately 1% ofthat from the overproducer used in our purification (15). Taking this into account, and also the final yield of protein from the number of cells used for the purification, it can be calculated that wild- type E. coli contains approximately 200 TagI molecules per cell. During preparation of this manuscript, Sakumi et al. (31) have reported an alternative method for purification to homogeneity of 3-methyladenine DNA glycosylase I from E. coli.

ACKNOWLEDGEMENTS This work is part of a project (No. D.68.90.018) supported by the Norwegian Research Council for Science and the Humanities (NAVF). Purified T4 denV enzyme was the generous gift of Dr. Priscilla Cooper. We thank Dr. Mutsuo Sekiguchi for communicating results prior to publication.

Abbreviations: AP, apurinic/apyrimidinic; FPLC, fast protein liquid chromatography.

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