143

Mutation Research, DNA Repair, 255 (1991) 143-153 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 0921877791000895 MUTDNA 06452

Drosophila methyltransferase activity and the repair of alkylated D N A Sami N. Guzder

a,

Mark R. Kelley b and Walter A. Deutsch

a

a Department of Biochemistry, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.) and b Department of Biochemistry and Molecular Biology Program, Loyola University Medical School, 2160 South FirstAvenue, Maywood, IL 60153 (U.S.A.) (Received 29 January 1991) (Revision received 19 March 1991) (Accepted 20 March 1991)

Keywords: DNA repair; Alkyltransferases; (Drosophila melanogaster); Alkylated DNA, repair; O6-Methylguanine

Summary The biochemical mechanism and developmental expression for the repair of alkylated DNA has been characterized from Drosophila. As in other organisms, the correction of O6-methylguanine in Drosophila was found to involve the transfer of a methyl group from DNA to a protein cysteine residue. Two methylated proteins witl~ subunit molecular weights of 30 kDa and 19 kDa were identified following incubation with [3H]-methylated substrate DNA and denaturing polyacrylamide gel electrophoresis. Identical molecular weights were found for the unmethylated forms of protein through their reaction to an antibody prepared against the 19 kDa Escherichia coli methyltransferase. Both Drosophila proteins are serologically reactive in adult males and females and most of the other developmental stages tested, with embryos representing the possible exception. The Drosophila proteins do not appear to be induced by sublethal exposures to alkylating agent.

When DNA is exposed to monofunctional alkylating agents such as N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU), the major modifications N 7methylguanine (N7mG), N3-methyladenine (N3mA), and O6-methylguanine (O6mG) are formed (Lawley and Thatcher, 1970). In the absence of DNA repair, O6mG can give rise to G : C to A : T transition mutations by anomalous base pairing during DNA replication (Hall and

Correspondence: Dr. Walter A. Deutsch, Department of Biochemistry, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.), Tel. 504-388-5148.

Saffhill, 1983; Loechler et al., 1984). The presence of N3mA, on the other hand, leads to cell killing known to be associated with these potent mutagens and carcinogens. To counter the mutagenic effects of alkylating agents, Escherichia coli depends upon an inducible protein that covalently transfers the methyl group from the 0 6 position of guanine in alkylated DNA to one of its own cysteine residues, which is accompanied by the irreversible loss of activity (Olsson and Lindahl, 1980). The purification of this inducible methyltransferase originally revealed a molecular weight of 19 kDa (Demple et al., 1982), although subsequent tests on the repair of other O-methylated products, particularly methyl phosphotriesters, concluded that in

144

fact a 39-kDa protein was involved (McCarthy et al., 1983; Weinfield et al., 1985). The gene encoding the 39-kDa polypeptide has been cloned (Sedgwick, 1983), in which its protein product reveals two domains of activity (Teo et al., 1986). The C-terminus contains a cysteine residue that is active in the repair of O6mG and O4mT, whereas the N-terminus contains a cysteine residue for the transfer of a methyl group from phosphotriesters. This latter transfer event was found to be critical for turning the methyltransferase protein into a transcriptional activator of the ada regulon which encodes not only the methyltransferase, but three other genes as well. One of these, alkA, encodes an inducible DNA glycosylase which is active against a variety of N-alkyl modifications, some of which are responsible for the cell killing described for alkylating agents. A question as to whether the 19-kDa and 39-kDa methyltransferase are both active in vivo has been resolved by learning that the 19-kDa species is only generated upon cell lysis, presumably by the proteolytic action of the ompT gene product (Sedgwick, 1989). However, E. coli do possess a noninducible, constitutively expressed 19-kDa methyltransferase protein, which is the product of the ogt locus and repairs O6mG and O4mT, but not methylphosphotriesters (Margison et al., 1985; Potter et al., 1987). A noninducible DNA glycosylase also exists that is specific towards NSmA and is encoded by the tag gene (Riazuddin and Lindahl, 1978). A methyltransferase activity against O6mG has been identified in a variety of eukaryotic cells (Bogden et al., 1981; Waldstein et al., 1982, Yarosh et al., 1984). These proteins seem to share some of the same characteristics of the Ada protein, especially the transfer of the methyl group from the O6-position of guanine to a cysteine residue. The eukaryotic methyltransferase proteins, however, are apparently more similar to the E. coli Ogt protein than the Ada protein since they fail to repair methylphosphotriesters present in alkylated DNA (Brent et al., 1988; Koike et al., 1990). Other modifications produced by alkylating agents, such as NVmG and N3mA, are repaired by DNA glycosylase activity(s) in cultured human lymphoblasts (Singer and Brent,

1981). A similar activity against N7mG has also been found in rodent livers (Margison and Pegg, 1981). Studies in Drosophila have previously shown that the three major alkylated bases in DNA appeared to undergo some form of DNA repair that was independent of DNA glycosylase activity. The loss of methyl groups could however be traced to a protein moiety (Green and Deutsch, 1983), although the exact transfer mechanism was not reported. We now show that in fact DNA methyl groups are transferred to a cysteine residue. Further, this paper also follows the developmental expression of transferase activity using an antibody prepared against the E. coli 19-kDa methyltransferase. Notably, serological cross-reactivity was virtually absent in embryos, consistent with our previous inability to detect biochemical activity at this stage of Drosophila development (Green and Deutsch, 1983). Materials and methods

Partial purification of methyltransferase activity from pupae. D. melanogaster (Oregon-R)embryos with an average age of 4 h were collected. The preparation and transfer of embryos to sterile, dead yeast-sucrose medium containing streptomycin, penicillin (25000 units/ml) and proprionic acid to inhibit yeast and bacterial growth has been described previously (Deutsch and Spiering, 1982). Pupae (190-240 h) were collected, washed with 0.01% Triton X-100/0.7% NaC1 and then with deionized water and stored at - 7 0 ° in 50 mM Tris-HC1, pH 7.5/2 mM E D T A / 5 mM DTT/Aprotinin (0.6 Trypsin Inhibitor Units (TIU)/ml buffer). Pupae were thawed on ice, and after removal of the storage buffer, homogenized in a 7-ml Dounce homogenizer. The homogenate was sonicated with two bursts for 30 sec each using a Branson sonicator at 4 A. The sonicate was centrifuged in a Beckman JA-20 rotor at 12000 rpm for 15 min at 4°C. The supernatant was filtered twice through a sterile Nitex screen and stored at 4 °C (Crude extract). For partial purification of the methyltransferase proteins, ammonium sulfate was slowly added to the crude extract to a final concentration of 50% w/v. After stirring for 30 min at

145

4 ° C, the solution was centrifuged at 13 000 rpm in a JA-20 rotor for 15 min at 4 ° C. The 50% supernatant was saved on ice. The protein pellet was resuspended in 2 ml of buffered 30% ammonium sulfate solution (20 mM Tris-HC1, pH 8.2/10 mM D T T / 3 mM EDTA/Aprotinin (0.6 TIU/ml)/ammonium sulfate), dispersed with a sterile pestle and stirred for 30 min at 4 ° C. After centrifugation, the 30% supernatant was retained and stored at 4 ° C. The protein content of all fractions was determined using the Bradford assay reagent.

Alkylation of calf-thymus DNA. Calf-thymus DNA (Type 1; Sigma) was dissolved in TE buffer (10 mM Tris-HCl, pH 8.2, 0.1 mM EDTA) to give a final concentration of 2-3 mg/ml and then mixed with 0.2 mCi [3H]N-nitrosourea (MNU, 1.75 Ci/mmole; Amersham). The final concentration of Tris was adjusted to 0.1 M and incubated at 37°C for 12 h. The DNA was then precipitated using 0.5 M LiCI and 2 v / v ethanol. The DNA pellet was washed with water-saturated ether, dried, resuspended in TE and dialyzed extensively against the same buffer. This procedure resulted in alkylated DNA with a specific activity between 700 and 2300 cpm//.~g DNA (0.36-1.2 pmoles//xg DNA). Methyltransferase assay. The assay for methyltransferase (MT) activity was essentially as described by Demple et al. (1983). The reaction mixture (0.12-0.24 ml) consisted of 50 mM TrisHCI, pH 8.2 (on occasion 20 mM Hepes-NaOH, pH 8.1, was substituted), 5 mM DTT, 2 mM EDTA and 0.004-0.01 mg labeled, alkylated calf-thymus DNA (CT-DNA; Green and Deutsch, 1983). Protein (0.2-3 rag) was added to the reaction buffer and incubated at 37 ° C for 30-40 min. Acetylated bovine serum albumin (AcBSA) was added in concentrations identical for pupal proteins, and served as a control for trapping of radioactivity in the protein pellet. Reactions were terminated by the addition of sodium acetate (pH 5.5) to 0.3 M and 2.5 vol of ethanol at - 2 0 ° C. The contents were mixed thoroughly and allowed to stand at - 7 0 ° C for 3-4 h, and then centrifuged for 15 min at 11000 rpm at 4°C. The ethanol supernatant was divided into two equal

parts, one for determining radioactivity, and the other for analysis by HPLC of alkylated base modifications. The remaining protein-DNA pellet was resuspended in 0.1 N HC1 (0.2-0.4 ml) and heated at 70 °C for 40 min and then cooled on ice. This treatment labilizes the major Nmethylated and some O-methylated lesions in the DNA substrate that remain after reaction with the MT protein. The mixture was then centrifuged, as above, and the HCl-supernatant saved for HPLC analysis and determination of radioactivity. The remaining pellet was solubilized by the addition of 0.1 M Tris-HCl, pH 8.2/5% SDS and heating at 70 °C for 30-40 min. The solubilized mixture was mixed with aqueous fluor and analyzed for radioactivity by scintillation counting.

High-performance liquid chromatography. Base modifications of MNU-treated DNA were resolved using a Partisphere 10 SCX column (Whatman) employing a linear gradient from 0 to 9 min of solution I (0.02 M sodium acetate, pH 4.0/5% methanol) to solution II (0.1 M sodium acetate, pH 4.0/30% methanol), with subsequent isocratic elution with Solution II for an additional 5 min. Authentic modified bases (Fluka) were used as markers. Fractions (0.5 min) were collected and mixed with aqueous scintillation fluor and radioactivity determined (counting efficiency of 40-50%).

Separation of proteins after a methyltransferase reaction. Reaction mixtures were subjected to Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) using a 12% gel, with molecular weight standards run in the same gel. Electrophoresis was carried out at 30 mA until the dye front just reached the bottom of the gel. Individual sample lanes were cut into 2-mm slices and placed into scintillation vials. The recovery of radioactivity associated with the methyltransferase protein(s) was determined by emulsifying the gel slice in a solution of Scintelene (Fisher)/5% Protosol (New England Nuclear), and heating the vial at 60 °C for 18 h. The vials were cooled overnight and radioactivity determined. A profile of the radioactivity associated with each sample, minus an AcBSA control, was drawn. A peak of radioactivity was indicative of a

146

putative methyltransferase protein, and its molecular weight estimated by comparing the distance migrated in the gel with respect to known molecular weight standards.

Induction of methyltransferase activity in third instar larvae. D. melanogaster embryos with an average age of 4 h were collected and transferred to dead yeast-sucrose medium as described previously (Deutsch and Spiering, 1982). Embryos were allowed to develop at 25 °C to third instar larvae (180-190 h). A sterile 30% sucrose solution was added to the bottle, whereupon the larvae floated to the surface and collected. The larvae were washed extensively with deionized water and then transferred to media bottles (100 ml) containing a piece of sterile Whatman 3 MM paper soaked in 5 ml methionine-deficient media (Robb, 1969), containing either 0.6 pM or 6 pM MNNG. Non-treated controls received 5 ml of Robb's media. The larvae were exposed to the mutagen for 6 h at 25 °C and then removed from the media. The larvae were subsequently washed with deionized water and stored at - 7 0 °C in 20 mM Hepes-NaOH, pH 8.1/2 mM E D T A / 5 mM DTI'. Concentrations of alkylating agents used had been extensively studied on the basis of toxicity and the ability to induce new proteins (Guzder and Deutsch, in preparation). Determination of the end product of the methyltransferase assay on MNNG-treated and nontreated third instar larvae. The MNNG-exposed and non-treated larvae were thawed on ice and homogenized in 1.5 v / v of cold high salt homogenization buffer (20 mM Hepes-NaOH, pH 8.1/10 mM D T T / 4 mM EDTA/0.4 M NaCI/0.005% Chaps/Aprotinin (0.6 T I U / m l buffer), 0.01 mM PMSF/1 mM leupeptin) in a sterile dounce homogenizer. After centrifugation at 11000 rpm for 15 min at 4 ° C the supernatant was filtered through a sterile Nitex screen and the filtrate assayed for methyltransferase activity. Reaction mixtures (0.24 ml) contained 20 mM Hepes-NaOH, pH 8.1, 5 mM DTT, 2 mM EDTA, 5% glycerol, crude extracts (1.1 rag) and labeled, alkylated CT-DNA. Incubations were for 40 min at 37 ° C. To determine that the radioactive methyl group had indeed been transferred to a cysteine

residue, assay mixtures were treated with proteinase K (0.1 mg) and aminopeptidase M (0.01 mg) for 12-14 h at 37 °C. The undigested proteins were precipitated with 0.1 M sodium acetate (pH 5.5)/2.5 v/v ethanol at - 7 0 °C for 4 h and centrifuged. The ethanol supernatant was evaporated to a final volume of 0.04 ml, and authentic S-methylcysteine and S-methylcysteine sulfone markers were added. The samples were applied to Whatman 3 MM paper and the products separated by descending chromatography for 16 h using propanol: deionized water (7 : 3). The chromatogram was dried and the markers visualized by spraying with a ninhydrin solution (0.3 g in 100 ml n-butanol/3 ml acetic acid). The spots in sample lanes which comigrated with authentic markers were excised and eluted with deionized water for 12 h, mixed with aqueous fluor (National Diagnostics), and radioactivity associated with these samples determined. Random sampiing of other areas of the chromatogram indicated radioactivity only migrating with the authentic makers.

Immunodetection of proteins in MNNG-exposed third instar larval extracts. Control, MNNG-exposed larval extracts (0.5 rag) and a homogeneous preparation of the 19-kDa methyltransferase protein of E. coli (0.0025 mg; from B Demple, Harvard University) were extracted with acetone (1 ml) at - 2 0 ° C. The precipitated Drosophila proteins were resuspended by brief sonication in a solution (0.09 ml) containing 0.1 M sodium carbonate/0.1 M DTT. Laemmli sample buffer (Laemmli, 1970) was added and the mixture boiled for 4 rain. These samples, along with standard molecular weight markers, were electrophoresed at 250 V on a 7-15% gradient polyacrylamide gel containing 0.1% SDS, until the dye-front just reached the bottom of the gel. The gel was then soaked in 25% isopropanol/0.05 M Tris-HC1, pH 7.5 for 20 min and then placed on a nitrocellulose (NC) membrane sandwiched in an electroblotting cassette (BioRad). The proteins in the gel were electroblotted on the NC membrane at 0.3 A for 3 h. The membrane was then placed in blocking buffer (10 mM Tris-HCl, pH 8.2/0.15 M NaCI/0.05% Tween - 2 0 / 5 % w / v non-fat powdered milk) and gently shaken overnight at

147

4 ° C. The NC membrane was then incubated with an antibody against the E. coli 19-kDa MT protein (kindly provided by Dr. L. Samson, Harvard School of Public Health), at a 1 : 2400 dilution for 30 min. The membrane was then washed 3 times with TBST (blocking buffer minus milk powder). The antigen: antibody complex was established by incubating with 125I-protein A (1 × 108 cpm). The membrane was washed 4 times with TBST, air-dried and exposed to XAR film (Kodak) at - 70 ° C for 7 days.

resolving the products of a methyltransferase assay by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and measuring the radioactivity within the gel. This procedure led to the identification of two labeled proteins, with subunit molecular weights of 30 kDa and 19 kDa (Fig. 1A). A 2500

66K

45K36K 29K

19K 15K

2000 ¸

Developmental expression of methyltransferase proteins in Drosophila. The expression of the methyltransferase proteins through the various developmental stages in Drosophila was followed using the Western Blot procedure and employing an antibody directed against the 19-kDa methyltransferase protein from E. coli. Crude extracts (0.5 mg) from embryos, larvae, pupae, and adults were subjected to electrophoresis on a 7-15% gradient polyacrylamide gel (Laemmli, 1970). A crude extract from HeLa cells (0.5 mg) was also electrophoresed with the above samples along with molecular weight standards until the dyefront just reached the bottom of the gel. The proteins in the gel were then electroblotted onto a nitrocellulose membrane and probed with the E. coli methyltransferase antibody as described above; the antigen: antibody complex was established in this case, however, by incubating with an anti-rabbit IgG-alkaline phosphatase conjugate. In a similar experiment, Drosophila males and females were directly placed in a lysis buffer containing 10 mm Tris-HCl, pH 7.4, 1% SDS, 0.2% Triton-X 100, 0.2% MP-40, 5 mM EDTA, 5 mM EGTA, 2 mM PMSF, 2 / ~ g / m l leupeptin, 2 /zg/ml pepstatin, and 0.2 T I U / m l aprotinin. Samples were boiled for 5 min, centrifuged for 60 min at 180000×g, and 40 /~g of protein electrophoresed through a 12% SDS-PAGE. Results

Two species of methyltransferase exist in Drosophila. Radioactive methyl groups transferred to methyltransferases remain covalently bound to the protein. The number of active methyltransferase proteins can therefore be determined by

L U

1500 1000 500 0

i0

20

30

40

50

Gel Slice B 25O0

66K

45K 36K 29K

÷

++

÷

19K 15K

+

+

Z~

IE 1500 I000 5OO 0q

lo

20 3o' Gel Slice

4o-

~o

C 2500. 2O00L U

1500 I000 500.

o6

-.

,~--

~0.~ 3.o

,fO~ ~

Gel Slice

Fig. 1. Radioactivity associated with proteins separated by SDSPAGE. Individualfractions(2 mg each) were incubatedwith aikylated DNA (3.1 pmoles [3H]-methyl groups) and then electrophoresed on a 12% polyacrylamidegel containing0.1% SDS. Crude, Panel A; 30% ammoniumsulfate,Panel B; 50% ammonium sulfate, Panel C. Molecularweight markers are shownat the top.

148 TABLE 1 PARTIAL FRACTIONATION OF METHYLTRANSFERASE ACTIVITY FROM DROSOPHILA PUPAE Expt.

Addition a

DNA rendered ethanolsoluble b

3H-Bases liberated by acid treatment b

Total meth yl GRPS repaired

HPLC analysis c NTmG(%) d O6mG(%)

N3mA(%)

(pmoles)

(pmoles)

(pmoles)

(pmoles)

(pmoles)

(pmoles)

1

None Crude extract

0.4 1.5

6.2 3.8

2.4

4.1 2.1(48)

0.6 0.2(66)

0.4 0.2(50)

2

None 50% NH4SO 4 extract 30% NH4SO 4 extract

1.2 0.4 0.4

9.4 5.5 5.4

3.9 4.0

6.2 2.1(66) 2.6(58)

0.8 < 0.1(90) < 0.1(92)

0.6 0.5(20) 0.5(20)

a Reactions contained an original 12 pmoles of 3H-DNA substrate for Expt. 1 and 18 pmoles for Expt. 2. Proteins were as follows: Crude, 2 mg; 50% and 30% ammonium sulfate fractions, 0.22 mg. b Reactions were terminated by ethanol precipitation as described in Materials and Methods. The remaining pellet was treated with 0.1 N HCI to liberate the major methylated bases, which were subsequently separated by HPLC. c The elution patterns are shown in Fig. 2. Recoveries were ordinarily around 50% of that applied to the column. d The percent methylated bases repaired as determined from control samples that were not exposed to Drosophila extracts.

Using ammonium sulfate precipitations, a protein of roughly 30 kDa appears in the supernatant of a 50% fractionation (Fig. 1C), whereas

an ammonium sulfate back extraction (30%) of that protein pellet yields a protein of approximately 19 kDa, with others possibly existing in

r

5

A

B

4 N 7 o

E o.

I0

I

I

20 Fraction r

I

I0

30

3O

20

Fraction I

I

1

I

I

r

I

I

5 13

C

4 -6 3~

--

3

E o. 2

,

-

I0

20 Froction

30

10

~

20

30

Fraction

Fig. 2. Separation of 3H-labeled alkylated bases by HPLC. Incubations containing labeled, alkylated calf-thymus DNA and Drosophila extracts were terminated with ethanol. The remaining protein-DNA pellet was resuspended in 0.1 N HCI, heated and the supernatant analyzed by HPLC. Repair of individual bases is provided in Table 1. Minus Drosophila extract (Panel A) and Crude (Panel B), Expt. 1; Minus Drosophila extract (Panel C) and 30% ammonium sulfate (Panel D), Expt. 2.

149 the molecular weight range of approximately 15kDa (Fig. 1B).

Drosophila pupae lack detectable DNA glycosylase activity toward alkylated DNA. D N A glycosylases act by hydrolyzing the D N A sugar-base N-glycosylic bond, liberating the free, modified or nonconventional base, and leaving a baseless site in the DNA. Only a small proportion of alkylated D N A is rendered ethanol soluble by Drosophila pupae (Table 1), and ordinarily could be accounted for by the presence of ethanol-soluble oligonucleotides (not shown), not the free base which would reflect authentic D N A glycosylase activity. The D N A that remained after ethanol precipitation was treated with 0.1 N HCI to liberate the methylated purines left intact after incubation with Drosophila protein. These acid-soluble samples were then analyzed by HPLC, and the amount of radioactivity co-migrating with authentic markers compared to samples not exposed to Drosophila extracts (Table 1 and Fig. 2). The results firmly establish the repair of O6mG, in which ammonium sulfate fractions removed over 90% of the radioactive methyl group associated with this D N A adduct. Notably, significant losses of radioactivity associated with NTmG, and to a lesser extent N3mA, were also observed, but as to whether they are subject to the same form of repair as O6mG remains unknown.

Recovery of S-[3H]methylcysteine. An important step toward placing the Drosophila methyltransferase proteins among those previously characterized was proving that the transfer of the labeled methyl group from alkylated D N A resulted in the production of S-[3H]methylcysteine. As part of this analysis, organisms exposed to low levels of an alkylating agent were also examined with an objective towards learning if not only methyltransferase proteins, but also D N A glycosylases, could be induced by such treatments. Since pupae are ordinarily found on the vertical face of media bottles, and therefore would not come in direct contact with the mutagen, we switched to treating third instar larvae, in which the biochemical levels of the in vitro methyltransferase activity are roughly one-third that observed

TABLE 2 QUANTITATION OF S-[3H]METHYLCYSTEINE PRODUCED BY THE TRANSFER OF 3H-METHYLGROUPS FROM DNA TO A PROTEIN MOIETY Sample

Control Treated larvae, 0.6 pM MNNG Treated larvae, 6.0 pM MNNG

3H-Methyl group in DNA (pmoles) 4.6

Recovery of S-[3H]Methylcysteine (pmoles) 1.6

4.3

2.0

4.6

1.6

a

a Values presented represent a combination of authentic Smethylcysteine and its oxidation product, S-methylcysteine sulf0ne. Samples taken of the protein pellet subsequent to HCI precipitation indicated that roughly 1 pmole of 3Hmethyl groups had been transferred to a protein moiety in the control and larvae treated with 6.0 MNNG, whereas 2 pmoles of 3H-methyl groups were recovered in the protein pellet of larvae that had been exposed to 0.6 pM MNNG. for pupae (not shown). Nevertheless, significant levels of S-methylcysteine were formed in a reaction combining alkylated D N A and third instar larvae (Table 2), thus confirming that in this regard it acts similarly to other methyltransferases. A preliminary attempt at inducing the levels of methyltransferase activity unfortunately did not meet with a great deal of success (Table 2). Furthermore, H P L C analyses of alkylated D N A exposed to extracts of both treated and nontreated organisms failed to reveal detectable levels of D N A glycosylase activity (not shown).

Immunodetection of methyltransferase proteins. Another method of identifying the methyltransferase proteins in Drosophila extracts utilized the Western Blot procedure. This was carried out on untreated and MNNG-exposed third instar larval extracts probed with an antibody originally generated against the E. coli 19-kDa methyltransferase protein (Teo et al., 1986). Interestingly, the polyclonal antibody appeared relatively specific for Drosophila proteins, with cross-reactivity identified in the region of 30 kDa and 19 kDa in both the nontreated control and MNNG-exposed third instar larval extracts (Fig. 3). As expected, the

150 Control

O.6pM

G.OpM

E.coli MT

Fig. 3. Antibody cross-reactivity to third-instar larval extracts. Proteins were separated by SDS-PAGE, electroblotted onto nitrocellulose, reacted with the antibody to the E. coli 19-kDa methyltransferase and developed with [12SI]protein A. The purified 19-kDa E. coli methyltransferase was run in the same gel with nontreated third instar larvae and larvae exposed to 0.6 pM and 6.0 pM MNNG. The entire gel is shown, with the top band at a molecular weight of 30 kDa, the bottom at 19 kDa.

homogeneous 19-kDa methyltransferase protein from E. coli also showed strong cross-reactivity, although it appears some degradation of the protein has taken place. Densitometric analysis of the autoradiograph showed that levels of both Drosophila proteins remained relatively unchanged in organisms exposed to the alkylating agent MNNG, which agrees with our previous biochemical analysis. The apparent specificity of the antibody reaction offered a means for monitoring the presence of the methyltransferase proteins for some of the more predominant stages of Drosophila development. Crude extracts were electrophoresed through a SDS-polyacrylamide gel (SDS-PAGE), and the antigen:antibody complex established in this case by anti-rabbit IgG-alkaline phosphatase conjugate, which provided a superior test for the presence of the 19-kDa methyltransferase as opposed to the use of 125I-protein A (Fig. 3); unfortunately, serological detection of the 30-kDa methyltransferase by this technique was not reproducible. As seen in Fig. 4, the presence of the 19-kDa methyltransferase was established for each of the developmental stages tested, with the

....

Helo Adults

Pupoe 5rd Instor 1st. Instar

Embryos Fig. 4. Developmental expression of the 19-kDa Drosophila methyltransferase. Samples (0.5 mg) from Drosophila and a HeLa cell extract (0.5 mg) were treated as described in Fig. 3, except the antigen:antibody complex was located with an alkaline phosphatase conjugated second antibody.

151 Discussion

Fig. 5. Expression of the 30-kDa and 19-kDa methyltransferases in Drosophilamalesand females.Conditionswere the same as in Fig. 4, except extracts were prepared in the presence of numerousprotease inhibitors(see Materials and Methods).Antibodycross-reactivityto maleprotein extractsis shownat the left, femalesto the right.

possible exception of the embryonic stage of development. Interestingly, the antibody also failed to combine with proteins extracted from HeLa cells, similar to that reported by Ceccoli et al. (1988) using independently derived polyclonal antibodies. Also evident in Fig. 4 is that some developmental stages contain antibody-positive proteins in addition to the 19-kDa species. This is especially true for adults. Since we considered this most likely due to proteolytic degradation, a separate experiment was conducted in the presence of a number of protease inhibitors. We were also additionally interested as to whether the methyltransferase was expressed in both sexes of Drosophila, and as a result tests were performed on both males and females. In this case, areas of antibody cross-reactivity were indeed detected in both sexes (Fig. 5), at molecular weights of 30-kDa and 19-kDa. It therefore appears that the 30-kDa protein can be protected from degradation through the use of a battery of protease inhibitors.

Among the organisms studied, Drosophila appear to contradict some of those expectations generally acknowledged for the repair of alkylated DNA. For example, both prokaryotes and other eukaryotes contain two DNA-repair pathways directed toward both the promutagenic and potentially lethal lesions produced by alkylating agents. One pathway appears to be absent in Drosophila, and normally involves the action of DNA glycosylases that liberate modified or nonconventional bases from DNA associated with the lethal consequences of DNA alkylation. Mutagenesis by these agents is prevented on the other hand by suicidal repair proteins that transfer the alkyl group to a cysteine residue residing within the protein. The Drosophila activity appears to carry out a similar self-methylating reaction that conceivably could act on a broader spectrum of alkylation damage than that characterized for other organisms since it is clear that we cannot explain the loss of methyl groups from N-modifications except by some form of transfer to a protein moiety. Based upon their recovery from SDS-PAGE, two species of Drosophila methyltransferase appear to be active in removing methyl groups from alkylated DNA. A question which remains unanswered is whether the 19-kDa protein is the proteolytic product of the 30-kDa methyltransferase, or for that matter the product of possibly even a larger molecular weight precursor. Studies are underway to address this problem, and if indeed these proteins are found related through proteolysis of a precursor, it would more closely resemble the Ada protein than the product of the ogt locus in E. coli, since the former, but not the latter, undergoes proteolytic cleavage (Potter et al., 1987). Alternatively, Drosophila may contain multiple molecular species of methyltransferases such as that found in Bacillus subtillis (Morohoshi and Munakata, 1987). In this case, however, both inducible and constitutive forms of protein have been identified, whereas in Drosophila it appears that neither species of protein are inducible. The developmental expression of Drosophila methyltransferases was traced by serological and,

152 to some extent, biochemical techniques. Notably, embryos a p p e a r to lack biochemical activity (Green and Deutsch, 1983), and to a large extent fail to show substantial amounts of antibody cross-reacting material, especially when compared to other developmental stages. The concurring biochemical and serological results found in embryos therefore suggests that the antibody is indeed detecting authentic methyltransferase proteins. As such, it is clear that the 19-kDa protein is present in all stages of development beyond embryos. The detection of the 30-kDa protein on the other hand was somewhat unpredictable, but nevertheless appears to be present, at the very least, in late larval stages of development, p u p a e and adults. Thus, most developmental stages of Drosophila contain both species of methyltransferases, and in pupae, both are active in alkylation repair. It is important to note that apparently adults contain proteins capable of repairing alkylated DNA. This does not a p p e a r to be the case for another repair enzyme, in which an a p u r i n i c / apyrimidinic endonuclease could only be confidently detected in the female germ line, leading us to speculate that perhaps D N A repair was absent in adult males (Venugopal et al., 1990). While this still might hold true for nucleotide excision repair in general, it certainly does not a p p e a r to be the case for the direct reversal of D N A damage by methyltransferases. On the other hand, since adult tissues are for the most part terminally differentiated, the presence of these proteins may not solely exist for D N A repair, although cells undergoing meiosis might warrant such attention. Attempts are now underway to purify the methyltransferase proteins from adults. Thus far, preliminary results indicate the 19-kDa protein is tightly associated with nuclear matrices, and could perhaps provide a means for obtaining a homogeneous preparation that has so far failed using techniques established for the isolation of the bacterial methyltransferase (Demple et al., 1983). On the other hand, the apparent specificity of the antibody used in this study offers a straightforward means toward the molecular cloning of the Drosophila methyltransferase gene(s), allowing a

direct route for the subsequent characterization of the encoded proteins.

Acknowledgements Our thanks go to Dr. Leona Samson for providing the antibody p r e p a r e d against the Escherichia coli 19-kDa methyltransferase. This work was supported by the Louisiana Agricultural Experimental Station (W.A.D.), a grant from N I H (W.A.D.), American Cancer Society G r a n t No. NP-674 (M.R.K.), and a Schweepe Career Development Award (M.R.K.).

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