The Escherichia coli 0"-Methylguanine-DNA Methyltransferase Does Not Repair Promutagenic 0"-Methylguanine Residues When Present in Z-DNA*

The repair of 0"-methylguanine present in N-meth- ylnitrosourea (MNU)-treated alternating polynucle- otides (MNU-poly(dG-dC)*poly(dG-dC) and MNU-poly(dG-me6dC)*poly(dG-me6dC)) was investigated us- ing 0"-methylguanine-DNA methyltransferase purified from Escherichia coli. Both modified polynucleotides are equally good substrates for the DNA methyltransferase when they are in the B-form. The substrate properties of the MNU-treated polynucleotides do not differ from those of MNU-treated DNA. One of these modified polynucleotides, MNU-poly(dG-me6dC)*(dG-me6dC), can adopt the Z-conformation under physio- logical conditions. The conformational transition of the poly(dG-me6dC)*poly(dG-me6dC) from the B-form to the Z-form was monitored by the modification of its spectroscopic properties and by the specific binding of antibodies raised against Z-DNA. The 0'-methyl- guanine residues are repaired in MNU-poly(dG- me6dC)-poly(dG-me6dC) cpm/nmol, radioactivity, 06-Methylguanine-DNA Methyltransferase Assay-The standard assay measured the disappearance of O'-methylguanine from [3H] MNU-treated DNA (20). The reaction mixture (100 pl) contained 50 nmol of [3H]MNU-DNA (-0.1 pmol of-06-methylguanine) in 5 mM Hepes-KOH (pH 7.5), 5 mM dithiothreitol, 1 mM EDTA, 50 NaCl, and 0-0.5 units of 06-MeGua-DNA methyltransferase. Alter- natively, 3 nmol of [3H]MNU-poly(dG-dC) or 2 nmol of [3H]MNU-poly(dG-me6dC) were used as substrates (-0.1 pmol of 06-MeGua). The reaction was carried out at 25 "C for 10 min. The mixture was then supplemented with authentic markers (7-methylguanine and OB- MeGua) and 0.1 N HCl (final concentration). The resulting mixture was heated for 30 min at 70 "C, cooled and centrifuged in Eppendorf microtubes, and neutralized with NaOH, and the products were separated by high pressure liquid chromatography. Alternatively, the hydrolysates were filtered through GF/C filters (Whatman), and the radioactivity bound to the filters reflected the methyltransferase activity. This assay was used during the course of the purification. each peak of activity was further measured by high pressure liquid chromatography because of some false positive responses.

It is generally assumed that the major conformation of DNA in biological systems is B-DNA. An alternative conformation, termed Z-DNA, was recently described. Left-handed Z-DNA has been found to occur in fragments of natural DNA, supercoiling being a major factor to stabilize this conformation (for review see Refs. 1-4 and references therein). Immunological assays using antibodies to Z-DNA provide strong 3 Fellow of the Fondation pour la Recherche Medicale and Association pour la Recherche sur le Cancer, while on leave from the Institute of Biology, Rio  evidence for the presence of Z-fragments in chromosomes of two dipterian species (5, 6) and in the nuclei of mammalian cells (7,8). The biological role of Z-DNA is not yet known. However, the consequences of the presence of Z-DNA fragments can be evaluated from the ability of cellular proteins to use Z-DNA as substrate. Proteins which specifically bind to Z-DNA have been described (9). Interactions between Z-DNA and several proteins, RNA polymerases (10, l l ) , histones (12, 13), methylases (14)(15)(16), nucleases (3,15,16), and DNA-glycosylase (17) have been studied.
Upon alkylation of DNA by chemical carcinogens such as N-methylnitronitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine, the main reaction products are 7-methylguanine, 3-methyladenine, 06-methylguanine ( 06-MeGua'), and the phosphotriesters (18). Lesions introduced in DNA by alkylating agents are repaired either by a DNA alkyltransferase or by the sequential action of a DNA glycosylase and an apurinic/apyrimidinic endonuclease (for review see Refs. 18 and 19). We have shown that the imidazole ring-opened form of 7-methylguanine was not excised by the specific DNA-glycosylase when this lesion was present in poly(dG-me6dC) in Zform (17). So far, the mutagenic and/or carcinogenic properties of alkylating agents are correlated with the persistence of 06-methylguanine residues in DNA (18). Mammalian and bacterial cells contain a protein, the 06-methylguanine-DNA methyltransferase, which repairs the 06-alkylguanine by transferring the alkyl group to one of its own cysteine residues, restoring the guanine in DNA in a single step (for review see Ref. 19).
In this study, we measured the effects of the conversion of the B-to the Z-conformation of a polynucleotide containing 06-methylguanine residues, on Escherichia coli 06-MeGua-DNA methyltransferase activity. We report that 06-methylguanine is very poorly repaired, if at all, when the alkylated poly(dG-me6dC) is converted to the left-handed Z-form.

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and MNU-poly(dG-me6dC) were 100, 800, and 1400 cpm/nmol, respectively. OB-methylguanine accounted for 6, 14, and 11% of the total radioactivity, respectively. The reaction was carried out at 25 "C for 10 min. The mixture was then supplemented with authentic markers (7-methylguanine and OB-MeGua) and 0.1 N HCl (final concentration). The resulting mixture was heated for 30 min at 70 "C, cooled and centrifuged in Eppendorf microtubes, and neutralized with NaOH, and the products were separated by high pressure liquid chromatography. Alternatively, the hydrolysates were filtered through GF/C filters (Whatman), and the radioactivity bound to the filters reflected the methyltransferase activity. This assay was used during the course of the purification. However, each peak of activity was further measured by high pressure liquid chromatography because of some false positive responses.

Preparation of E. coli O'-Methylguunine-DNA Methyltransferase-
The protein was purified from E. coli BS 21 (adc thy his), a strain constitutive for the synthesis of the 06-MeGua-DNA methyltransferase (20). The cells were grown at 37 'C in LB broth to late exponential phase. Bacteria were centrifuged, washed, resuspended in lysis buffer (20 mM Hepes-KOH, pH 7.5,5 mM Na,EDTA, 5 mM dithiothreitol), and stored at -70 "C. The cells (5 g) were lysed using lysozyme as already described (21). All subsequent steps were performed at 4 "C. The lysate was sonicated to reduce viscosity and clarified by centrifugation (25,000 X g for 15 min). The supernatant (Fraction I) was precipitated by slow addition of an equal volume of 2% streptomycin sulfate in water (Rhone Poulenc, Paris). The suspension was centrifuged and the supernatant (Fraction 11) was precipitated by ammonium sulfate (Schwarz/Mann, enzyme grade) to 75% saturation. The precipitate was collected by centrifugation and the pellet was resuspended in buffer A (50 mM Hepes-KOH, pH 7.5,l mM EDTA, 1 mM 0-mercaptoethanol, 10% glycerol) and dialyzed against Buffer A containing 0.25 M NaCl (Fraction 111). This fraction was chromatographed on a column of DEAE-cellulose (DE52, Whatman) packed, equilibrated, and eluted with buffer A containing 0.25 M NaCl. The active fractions, which eluted after the bulk of proteins were pooled (Fraction IV) and further dialyzed against buffer B (15 mM potassium phosphate, pH 7.4, 1 mM 8-mercaptoethanol, 1 mM EDTA, 10% glycerol), were loaded onto a phosphocellulose column (P-11 Whatman). The column was washed with buffer B and eluted with buffer B containing 0.5 M NaCl. 06-MeGua-DNA methyltransferase activity coeluted with the peak of proteins. The active fractions were pooled and further dialyzed against buffer B containing 0.05 M NaCl. The phosphocellulose fraction (Fraction V) showed a specific activity of 200 units/mg under the assay conditions used. This 100-fold purified fraction was used in all the experiments reported here. One unit of enzyme was defined as the amount of protein needed to remove 1 pmol of 06-MeGua from MNU-treated calf thymus DNA at 25 "C in 10 min.
High Pressure Liquid Chromatography of Methylated Guunines-After hydrolysis, the methylated purines were separated and characterized by high pressure liquid chromatography. The column was a CI8 pBondapak (Waters), which was isocratically eluted at 1.5 ml/ min. The mobile phase was 50 mM NHhHzPO,, pH 4.5, and 10% methanol (v/v). The fractions were collected and the radioactivity was measured by liquid scintillation counting. The radioactive products were identified by coelution with internal markers. Under these conditions, 7-methylguanine and OB-methylguanine eluted at 7 and 12 min, respectively.
Conformational Transitions of Modified Polynucleotides-The conformational state of MNU-poly(dG-dC) and MNU-poly(dG-me6dC) was assessed by: (i) the absorption ratio Am/Aze~ (22), determined using a Zeiss PMQ3 spectrophotometer; (ii) the circular dichroism spectra, monitored using a computerized Jouan-Roussel Dichrograph IV (Ellipticities were expressed as At = t~ -CR. Peak molar extinction coefficient used was 7 X lo3 for poly(dG-me5dC).); and (iii) the precipitation of the MNU-treated polynucleotides in the presence of specific antibodies against Z-DNA (a generous gift of Drs. B. Malfoy and M. Leng, Centre de Biophysique Molkulaire, OrlCans, France) (23). For radioimmunoassay, 10 pl of the serum were added to each assay. The mixture was incubated at 37 "C for 20 min, cooled to 0 "C, and centrifuged at 10,OOO X g for 30 min. The radioactivity of the supernatant was determined; it is a measure of the fraction of the polynucleotide in the B-form.

RESULTS
Substrates containing 06-MeGua residues were obtained by treatment of calf thymus DNA, poly(dG-dC), and poly(dG-me5dC) with N-[3H]methyl-N-nitroso~rea. These modified polynucleotides were used as substrates for the purified 0'-MeGua-DNA methyltransferase from E. coli. Fig. 1 shows that Ofi-MeGua disappears from MNU-poly(dG-dC) incubated in the presence of 0'-MeGua-DNA methyltransferase. The rate of repair of the lesion in the polynucleotide is identical to that observed in MNU-DNA. The 06-MeGua-DNA methyltransferase activity is neither modified by addition of 4 mM MgCl, nor by heat treatment of MNU-poly(dG-dC) at 50 "C for 10 min in the presence of MgCl,, prior to being used as substrate for O'-MeGua-DNA methyltransferase (Fig. 1). Under these experimental conditions, even after heat treatment at 50 "C in the presence of MgCl,, the modified polymer is in B-conformation as judged by circular dichroism (data not shown).
Poly(dG-dC) cannot adopt the Z-conformation under conditions which are compatible with Ofi-MeGua-DNA methyltransferase activity. In order to measure the repair of 0'-MeGua in the same polynucleotide, either in the B-or in the Z-form, we used MNU-poly(dG-me'dC). This polynucleotide is in the B-conformation at low salt concentration in the absence of divalent cations (24). However, heat treatment of poly(dG-me5dC) in the presence of small amounts of MgCl, induces the transition from the B-to the Z-form (24). In our assay conditions, 50 mM NaC1, the MNU-poly(dG-me'dC) is in the B-form as indicated by its Am5/AZw ratio (Fig. 2, inset) and circular dichroism (Fig. 3), and it is substrate for the 0'-  MeGua-DNA methyltransferase (Fig. 2). It appears that there is a slight difference in the maximum level of activity that 06-MeGua-DNA methyltransferase can reach with MNUpoly(dG-dC) and poly(dG-me6dC) in the B-form (Figs. 1 and  2). This difference seems to be due to intrinsic properties of the polynucleotides rather than to the presence of a fraction of the MNU-poly(dG-me6dC) in the Z-form (Figs. 2 and 3). Figs. 2 and 3 show that addition of 4 mM MgClz or heat treatment alone of MNU-poly(dG-me6dC) prior to incubation with 06-MeGua-DNA methyltransferase, neither modifies the A295/&60 ratio, the CD spectrum, nor the activity of the 06-MeGua-DNA methyltransferase. Incubation of MNUpoly(dG-meSdC) for 10 min at 25 "C (which are the assay conditions for 06-MeGua-DNA methyltransferase) does not induce significant conversion, if any, to the Z-form, as judged by the Az95/A2W ratio or CD spectrum which remains unchanged (not shown). By addition of 4 mM MgC12 and heat treatment for 10 min at 50 "C, the MNU-poly(dG-me6dC) adopts the Z-conformation, as judged by the increase of the Am6/AZm ratio (Fig. 2, inset) and circular dichroism (Fig. 3). Fig. 2 shows that 06-MeGua residues are poorly repaired, if MNU-poly(dG-me6dC) is converted to the 2-form. The amount of 06-MeGua repaired in the left-handed polymer is at the threshold of the detection and is not modified by the addition of a large excess of protein (Fig. 2). The MNUpoly(dG-mesdC) in Z-conformation cannot be sedimented out of solution (data not shown) as described for Z*-poly(dG-dC) (25), suggesting the absence of intermolecular aggregation. Furthermore, the lack of activity of the 06-MeGua-DNA methyltransferase is not due to heat treatment in the presence of MgClZ, as MNU-poly(dG-dC) treated under the same conditions remains a good substrate (Fig. 1).
The kinetics of conversion of MNU-poly(dG-meSdC) to the Z-form in the presence of MgClz was measured at 37 "C. Fig.  4 (inset) shows that half of the transition is observed within 10 min, and it is completed after 30 min. Taking advantage of the very low, if any, conversion at 25 "C (not detectable within 10 min), the activity of 06-MeGua-DNA methyltransferase can be measured using as substrate the MNU modified polynucleotide containing increasing amount of Z-form. Fig.  4 shows that the kinetics of inactivation of the 06-MeGua-DNA methyltransferase is well correlated with the kinetics of conversion from the B-form to the Z-form. These results again suggest that 06-MeGua is not repaired by the OB-MeGua-DNA methyltransferase when present in polymer in the left-handed Z-conformation. The lack of activity is not due to an irreversible inactivation of the enzyme, as 06-MeGua-DNA methyltransferase incubated in the presence of a substrate in Z-form remains able to repair subsequently added substrate in B-form (not shown).
Drugs that intercalate in DNA, such as ethidium bromide, induce the transition from the Z-form to the B-form (26, 27).    5 shows that when MNU-poly(dG-me5dC) is in the Zform, the 06-MeGua is not removed from the polynucleotide, as already shown above. Upon addition of ethidium bromide, the complex ethidium bromide-MNV-poly(dG-me5dC) becomes substrate for the 06-MeGua-DNA methyltransferase in a cooperative manner. The transition was assessed using antibodies to Z-DNA, and the sigmoidal curve observed is identical to that obtained for 06-MeGua-DNA methyltransferase activity (Fig. 5). The amount of ethidium bromide required to induce 50% transition is 0.1 ethidium bromide molecule/nucleotide. When the amount of ethidium bromide is higher than 1 molecule/nucleotide, the 06-MeGua-DNA methyltransferase is inhibited, the template being either MNU-poly(dG-me5dC) in B-form (Fig. 5) or MNU-poly(dG-dC) (not shown). Furthermore, reversion from the Z-to the B-form can be induced by addition of EDTA (25). The addition of an excess of EDTA, relative to MgC12, to MNUpoly(dG-me6dC) in Z-form, completely restores the ability of the polymer to be a substrate for 06-MeGua-DNA methyltransferase (not shown). These two last results show a high correlation between the conditions which allow the Z-to-B transition and the recovery of the 06-MeGua-DNA methyltransferase activity.

DISCUSSION
The ability of DNA repair enzymes and/or proteins to maintain the genome's integrity is of major importance for the cell. So far, the initial step of the repair of DNA methylated by chemical carcinogens depends upon DNA-glycosylases and DNA-methyltransferases (18, 19, 21). In vitro experiments have shown that the imidazole ring-opened form of 7methylguanine is not repaired by the specific DNA-glycosylase when this lesion is in Z-DNA (17). We show that this ) was converted to the Z-form by pretreatment at 50 "C in the presence of 4 mM MgC12. Increasing amounts of ethidium bromide were added, and the substrate was further heated at 50 "C for 10 min. It was then used as substrate for O'-MeGua-DNA methyltransferase (M). As a control experiment, the same substrate was similarly processed, except that MgC12 was omitted (M). The Z 4 B transition was also assessed by immunoprecipitation, as described under "Experimental Procedures" (V-V).
The amount of radioactivity in the supernatant accounts for the amount of the polynucleotide in the B-conformation. holds true for the repair of 06-MeGua when in Z-DNA. This lesion is repaired by the direct transfer of the methyl group from the modified base to one of the cysteine residues of 06-MeGua-DNA methyltransferase. Therefore, two distinct classes of repair enzymes are not able to repair lesions when present in Z-DNA. Similarly, the HhaI methylase or restriction endonuclease (15, 16) are not able to act on DNA in the Z-form. These results demonstrate that neither the DNA lesion itself (Ref. 17 and this work) nor the primary base sequence (15, 16) are the only basis for the formation of proper substrate for these proteins. The structure of the helix plays also an important role, either on the recognition of the lesion or on the reaction. In order to increase the biological relevence of the effect described in this paper, it would be important to show that it remains true in natural DNA, where the B + Z transition would be driven by supercoiling.
Therefore, the lack of repair of 06-MeGua residues in Z-DNA may result in the persistence of such lesions in the cellular genome. As the mutagenic and carcinogenic potency of alkylating agents is reasonably associated with their ability to produce 06-MeGua and to the lack of repair of this lesion (28), one can propose that the formation of O'-methylguanine in DNA fragments which adopt the Z-conformation will increase the mutagenic effect of 06-MeGua at these sites. The physiological methylation of the cytosine residues in eukaryotic systems (29) could provide repeating sequences which could easily adopt the Z-conformation. These DNA fragments could display an unexpected high mutation rate after treatment with chemical carcinogens. This possibility was proposed to occur in N-acetoxy-N-2-acetylaminofluorene-induced mutation, as it is observed at a limited number of sites (30). The spontaneous mutagenesis might be also greatly increased if the enzymes which control the repair of mismatched bases (31) are similarly inhibited. Therefore mutational hot spots could be a consequence of Z-DNA conformation as previously suggested for quasi palindromic sequences (32). The occurrence of Z-DNA fragments in the genome could provide sequences with a high mutation rate, whereas the major part of the genome (in B-form) will display a low mutation rate. The hypothesis would imply an evolutionary function for Z-DNA.