Proc. Natl. Acad. Sci. USA Vol. 89, pp. 9331-9334, October 1992 Biochemistry

Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II (environmental carcinogenesis/mutagenesis/DNA modification/DNA repair/cyclic DNA adducts)

ZDENKA MATIJASEVIC*, MUTSUO SEKIGUCHIt, AND DAVID B. LUDLUM*t *Department of Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655; and tDepartment of Biochemistry, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan

Communicated by James A. Miller, July 6, 1992

ABSTRACT The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G -- A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno[14Clguanine-containing DNA substrate by nick-translating DNA with [14CldGTP and modifying the product with chloroacetaldehyde. E. coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein- and time-dependent manner. This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process. Similar enzymatic activity in eukaryotic cells might protect them from exposure to metabolites of vinyl chloride.

gated the activity of this enzyme towards an EG-containing DNA substrate. Both glycosylase II and 06-alkylguanine-DNA alkyltransferase are synthesized by E. coli in increased amounts as part of the adaptive response to methylating carcinogens (17). 06-alkylguanine-DNA alkyltransferase removes alkyl groups from the Q6 position of guanine and from phosphotriesters, thereby restoring the original structure. Glycosylase II acts in a different way, releasing modified bases from the DNA and leaving apurinic sites which are subject to further repair. Glycosylase II releases 3- and 7-alkylated purines as well as 02-methylcytosine and 02-methylthymine from DNA (17). The recent finding that N2,3-ethanoguanine is also released extends the specificity of this enzyme to DNA bases that bear exocyclic groups. The studies reported here show that the enzyme has activity towards a base that has been associated with vinyl chloride carcinogenicity.

The industrial chemical vinyl chloride is carcinogenic in animal tests and is associated with an increased incidence of hepatic angiosarcomas in exposed workers (1). Vinyl chloride is metabolized by the cytochrome P450 system in the liver to the unstable electrophile chloroethylene oxide, which rearranges spontaneously to the more stable alkylating agent chloroacetaldehyde (CAA) (2, 3). Chloroethylene oxide and CAA react with DNA to form a variety of adducts, many of which have been identified in vivo (4-10). The question has arisen, therefore, as to which DNA modification or modifications may be responsible for initiating the carcinogenic process. The modified base that is found in the greatest quantity in livers of animals exposed to vinyl chloride is 7-(2-oxoethyl)guanine (oeG) (4, 6, 7). This base does not cause misincorporation when it is present in a DNA template strand (11), but it does form apurinic sites, which could be mutagenic or carcinogenic. On the other hand, N2,3-ethenoguanine (EG) has been shown to mispair in in vitro transcription systems (12, 13) and causes G -- A transitions in Escherichia coli (14). These results suggest that EG may be of considerable biological significance. Cells could be protected against the carcinogenic effect of vinyl chloride if they possessed a mechanism which could remove eG from DNA. Although EG is a persistent lesion in rat liver (9), the literature contains a report that homogenates of a rat brain tumor cell line contain a glycosylase which releases EG as the free base (15). However, crude extracts were used in these studies. Recently, we have found that E. coli 3-methyladenine (m3Ade) DNA glycosylase II (glycosylase II) purified from the cloned gene releases the related DNA adduct N2,3-ethanoguanine from DNA modified by haloethylnitrosoureas (16). Accordingly, we have investi-

MATERIALS AND METHODS Materials. [3H]Methylnitrosourea (1 Ci/mmol; 1 Ci = 37 GBq) and [14C]dGTP (534 mCi/mmol) were purchased from Amersham. [3H]Thymidine-labeled E. coli DNA (23,100 cpm/jig) was obtained from Miles. eG was prepared as described by Sattsangi et al. (18); 06-methylguanine was allowed to react with CAA and the methyl group was removed by hydrolysis with 0.2 M HCO for 2 hr at 1000C. The product was purified to homogeneity by high-performance liquid chromatography (HPLC) on a C18 column. Marker oeG was a gift from James A. Miller (McArdle Laboratory for Cancer Research, Madison, WI). Calf thymus DNA came from Sigma; enzymes for performing nick-translation and purified glycogen were obtained from Boehringer Mannheim; and reagents for gel electrophoresis were from Bio-Rad. Preparation of DNA Substrate. Calf thymus DNA was radiolabeled in guanine by nick-translation with [14C]dGTP, specific activity = 534 mCi/mmol. The nick-translation mixture contained, in a total volume of 100 Al: 3 ug of DNA; 2.5 ACi of [14C]dGTP at 47 ,uM; unlabeled dATP, dCTP, and dTTP at 40 ,uM each; 30 units of DNA polymerase I; and 0.8 jug of DNase I. This mixture was incubated for 1 hr at 160C, and then the DNase I was inactivated by heating to 650C for 5 min. The reaction mixture was cooled to 16'C and radiolabeling was continued for an hour after addition of 10 units of DNA polymerase I. The reaction was stopped by heating to 650C for 5 min, and unincorporated nucleotides were separated on an NACS-52 Prepac column (BRL), using 0.2 M NaCl in 10 mM Tris.HCl/1 mM EDTA, pH 7.2, for DNA elution. Finally, the DNA was precipitated with ethanol; typically, the specific activity of the DNA was 2 x 105 Abbreviations: CAA, chloroacetaldehyde; glycosylase II, 3-methyladenine DNA glycosylase II; eG, N2,3-ethenoguanine; m3Ade,

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3-methyladenine; oeG, 7-(2-oxoethyl)guanine. tTo whom reprint requests should be addressed.

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cpm/pug. Since half of the [14C]dGTP radioactivity was in deoxyribose and the counting efficiency determined with [14C]hexadecane was 95%, the specific activity of the guanine base in nick-translated DNA was 175 cpm/pmol, assuming that radiolabeled deoxyguanylic acid had equilibrated with nonlabeled deoxyguanylic acid in the DNA. CAA was prepared as needed from CAA diethylacetal (chloroacetal). Equal volumes of chloroacetal and 2 M HCl were boiled together at 1570C for 10 min, and the reaction mixture was neutralized with 6 M NaOH/0.5 M sodium phosphate, pH 7.0 (1:1, vol/vol). Freshly prepared CAA was added in molar excess to [14C]guanine-labeled DNA in 0.5 M sodium phosphate, pH 7.0, and allowed to react for 20 hr at 370C as described by Oesch and Doerjer (8). Finally, CAAmodified, nick-translated DNA (CAA-[14C]G-DNA) was passed through an NACS-52 Prepac column and precipitated with ethanol. HPLC Analysis. Guanines released from CAA-[14C]GDNA were separated on an Alltech Spherisorb 5 gum (4.6 x 250 mm) C18 column eluted at 1 ml/min with increasing concentrations of acetonitrile in 25 mM KH2PO4, pH 6, as follows: 0% acetonitrile for 13 min, 0-20% acetonitrile over 10 min, and 20o acetonitrile for 20 min. The ultraviolet absorbance of the markers was monitored during the run at 270 nm with a Perkin-Elmer LC-55 spectrophotometer. Oneminute fractions were collected and dissolved in Ultima Gold (Packard Instrument), and their radioactivities were measured in a Beckman LS-1800 scintillation counter. The radioactive content of each fraction was plotted versus elution time, and the total activity in each peak was calculated by a computer program that automatically subtracts background. Purines were released from CAA-[14C]G-DNA by treatment with 0.1 M HCl for 16 hr at 37°C, or with 0.03 M phosphoric acid for 1 hr at 100°C. Both treatments resulted in the release of guanine and one new peak of radioactive material that coeluted with eG marker. This peak contained between 7% and 9% of the total radioactivity in different alkylations. As reported previously, no oeG was detected in this substrate (8). E. col Glycosylase H. Glycosylase II was purified to homogeneity from an E. coli alkA MS23 strain that carries the plasmid pYN1000, which contains the E. coli alkA+ gene (19). Purification through the phosphocellulose and DNAcellulose chromatography steps was performed as described previously (19). SDS/polyacrylamide gel electrophoresis of the product revealed a single band, which corresponded to the 30,000-dalton protein as expected. The enzyme had an activity of 2.4 units/mg; 1 unit was defined as the amount of enzyme that releases 1 pmol of m3Ade per min from [3H]methylnitrosourea-modified DNA, as described previously (16). This enzyme was tested for nonspecific nuclease activity by measuring its ability to release 3H from double-stranded [3H]thymidine-containing DNA or from this substrate after apurinic sites had been introduced by mild acid depurination (20). No nonspecific release of radioactivity was detected when 18,000 cpm of native or depurinated [3H]thymidinecontaining DNA was incubated with 0.7 unit of glycosylase II under assay conditions. Reaction mixtures used to determine the protein dependence of eG release contained in a total volume of 200 p1: 70 mM Tris HCI, pH 7.5; 10 mM EDTA; 1 mM dithiothreitol; 0.05 ,g (11,100 cpm) of CAA-[14C]G-DNA substrate with 6.3% eG; and various amounts of enzyme. The reaction mixture used to determine time dependence of eG release contained in 1 ml of the same buffer: 3.0 units of enzyme and 0.38 gg (76,250 cpm) of CAA-[14C]G-DNA substrate with 7.0%o eG. Aliquots (200 ul) were analyzed at 0, 5, 10, 20, and 40 min. Mixtures were incubated at 370C for the indicated time, and DNA was precipitated with ethanol in the presence of glycogen as carrier. The supernatant was dried under

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FIG. 1. HPLC profile on a C18 column of radioactivity released from CCA-[14C]G-DNA by acid depurination. Retention times for optical markers are indicated (v).

reduced pressure, and residues were redissolved in water and, after passage through a DEAE-Sephadex A-25 column (1-ml bed volume) to remove any oligonucleotides which might be present, were analyzed by HPLC. The identity of the eG released by the enzyme was confirmed by collecting the peak from the C18 column and rechromatographing it with the marker on an Applied Science Absorbosphere 5 pum (4.6 x 250 mm) SCX column eluted at 0.7 ml/min with 10 mM ammonium formate buffer, pH 3.9. Similarly, the identity of the unknown peak was investigated by cochromatography with known markers on the SCX column.

RESULTS As shown by the HPLC profile in Fig. 1, it was possible to obtain a satisfactory substrate by incorporating [14C]guanine into DNA by nick-translation and subsequently modifying a fraction of the guanines with CAA. This profile shows the presence of only one modified base, eG, in the fraction released by mild acid hydrolysis. Marker oeG eluted somewhat later than guanine, but before eG; there was no evidence for the presence of this derivative in the CAA-modified

substrate. Fig. 2 shows the results of a typical experiment in which CAA-[14C]G-DNA substrate was incubated with active or heat-inactivated enzyme. A large peak of radioactivity that

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FIG. 2. HPLC profile on a C18 column of radioactivity released from CCA-(14C]G-DNA after incubation for 2 hr at 37rC with 2.0 units of E. coli glycosylase II. (Left) Results with active enzyme. (Right) Results with heat-inactivated enzyme.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Table 1. HPLC retention times of optical markers and radiolabeled peaks Retention time, min SCX C18 column* columnt Compound Guanine 10.5 3.9 oeG 15.3 3.9 24.4 5.7 eG 25.0 6.0 [14C]EG 14C-labeled unknown 13.0 5.0 *Alltech Spherisorb 5 ,um (4.6 x 250 mm) C18 column eluted at 1 ml/min. tApplied Science Absorbosphere 5 jtm (4.6 x 250 mm) SCX column eluted at 0.7 ml/min.

coelutes with eG on this C18 column was released by the active enzyme. When this peak of radioactivity was collected and rechromatographed, it coeluted with an EG optical marker on an SCX column as shown in Table 1, confirming its identity as eG. In addition to the peak of EG, a small peak of radioactivity was consistently observed that eluted slightly after a guanine marker (see Fig. 2 and Table 1). (There is normally a 1-min lag between the position of the optical marker and the radioactivity in these profiles.) When this peak was collected, it eluted slightly after guanine on an SCX column. We have no further data as to its identity, although we believe it may be 1,N2-ethenoguanine or the hydrated form of EG as discussed below. Fig. 3 shows that the release of eG is dependent on the amount of glycosylase II added to the incubation mixture; approximately 10% of the eG present is released by 2 units of enzyme. Finally, Fig. 4 shows that eG is released steadily at a somewhat decreasing rate over a 40-min period. The initial rate of release of eG obtained from this figure is 0.03 pmol/min; by 40 min, 7.5% of the eG in the substrate had been released.

DISCUSSION Since radiolabeled CAA was not readily available, we prepared a suitable DNA substrate for repair studies by nicktranslation followed by reaction with unlabeled CAA. This procedure is general in its application and assists in the identification of adducts, since only derivatives of the radiolabeled base incorporated by nick-translation are detected. It is important to note, however, that [14C]dGTP rather than [3H]dGTP must be used in this procedure because 3H in the

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Glycosylase 11 (units) FIG. 3. Enzyme-dependent release of eG from CCA-[14C]GDNA by E. coli glycosylase II after incubation for 1 hr at 37°C.

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FIG. 4. Time-dependent release of eG from CCA-[14C]G-DNA after incubation with 0.6 unit of E. coli glycosylase II at 37°C.

8 position of deoxyguanosine is lost when the 7 position is alkylated (21). As shown in Fig. 1, only one derivative was detected in the bases released from CAA-[14C]G-DNA by mild acid treatment; oeG was specifically absent, as reported earlier (8). However, a small additional peak of radioactivity was released by glycosylase II as shown in Fig. 2. This peak appeared at 13 min in a position that is obscured by guanine in Fig. 1. The radiolabeled material in this peak consistently eluted approximately 2.5 min behind the guanine marker on the C18 column and did not coelute with either guanine or oeG on an SCX column, as shown in Table 1. Therefore, it apparently represents a new CAA-induced modification in DNA. Since CAA produces 1,N2-ethenoguanine when it reacts with guanosine (18), this peak of radioactivity could represent 1,N2-ethenoguanine. It could also be the hydrated form of eG, since both the hydrated and dehydrated forms of ethenoadenine and ethenocytosine are formed when CAA reacts with the corresponding polynucleotides (22). The data in Figs. 2-4 show clearly that eG is a substrate for glycosylase II; absence of nonspecific activity in this purified enzyme eliminates the possibility that the release is caused by nonspecific nuclease activity. By making the assumption that an equilibrium is reached between the radiolabeled and unlabeled guanine nucleotides during the nick-translation procedure, it is possible to calculate a specific activity for the guanine and eG in the substrate. Although the density of alkylation of the DNA substrate and other factors may affect rate, some comparisons can be made concerning the relative rates of release of m3Ade and eG by glycosylase II. The initial rate of release of eG by 0.6 unit of glycosylase II in Fig. 4 is 0.03 pmol/min; accordingly, 1 unit should release 0.05 pmol/min. Since the conditions used in this experiment are similar to those used in assaying the enzyme (1 unit of activity is defined as that amount of enzyme which releases 1 pmol/min of m3Ade), it would appear that glycosylase II is only 1/20th as active towards EG as it is towards

m3Ade. Nevertheless, this activity is probably important in preventing mutagenesis in bacteria, since EG is known to be a mutagenic lesion (14). Reports that human placenta contains a glycosylase capable of releasing ethenoadenine (23) and that brain tumor cells contain a glycosylase capable of releasing both ethenoadenine and eG (15) suggest that these glycosylases may be important defense mechanisms against carcinogenesis in higher organisms. Finally, in view of the mutagenic effects of eG and the known carcinogenicity of the haloethylnitrosoureas (24), it is interesting to compare the structure of eG with the structure

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Proc. NatL Acad. Sci. USA 89 (1992) N

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XJN

7.

N2,3-etheno

N2,3-ethano

guanine

guanine

(from VC)

(from CNU)

8. 9.

10.

FIG. 5. Structures of the two closely related exocyclic adducts of guanine which are released from DNA by E. coli glycosylase II. N2,3-ethenoguanine (eG) is found in DNA of animals exposed to vinyl choride (VC), and N2,3-ethanoguanine is a product of the reaction of N-(2-chloroethyl)-N-nitrosourea (CNU) with DNA.

11.

of N2,3-ethanoguanine, a DNA base modification caused by the haloethylnitrosoureas. These two DNA base adducts differ only in the saturation of the exocyclic ring, as shown in Fig. 5. Although saturation would also convert this ring from a planar to a puckered form, it is nevertheless interesting that two carcinogenic agents, vinyl chloride and the haloethylnitrosoureas, produce such similar DNA modifications. It is of further interest that both have now been found to be substrates for glycosylase II.

14.

The skilled laboratory assistance of Ms. Paula Ritchie is gratefully acknowledged. This work was supported by Grants CA-44499 and CA-47103 from the National Cancer Institute, Department of Health and Human Services.

19.

1. International Agency for Research on Cancer (1982) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans (Int. Agen. Res. Cancer, Lyon, France), Suppl. 7, 373-376. 2. Guengerich, F. P., Crawford, W. M. & Watanabe, P. G. (1979) Biochemistry 18, 5177-5182. 3. Bartsch, H., Malaveille, C., Barbin, A. & Planche, G. (1979) Arch. Toxicol. 41, 249-277. 4. Osterman-Golkar, S., Hultmark, D., Segerback, D., Calleman,

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