©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Metal Dependence of Transcriptional Switching in Escherichia coli Ada (*)

(Received for publication, July 27, 1994; and in revised form, January 5, 1995)

Lawrence C. Myers (1)(§) François Jackow (2) Gregory L. Verdine (1) (2)(¶)

From the  (1)Program for Higher Degrees in Biophysics and the (2)Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl transfer process is autocatalyzed by coordination of the acceptor residue, Cys, to a tightly bound zinc ion. Kinetic data reveal a 4-fold reduction in the methylphosphotriester repair activity for the Cd(II) form of Ada versus the native Zn(II)-bound form, thus confirming a direct role for the metal in autocatalysis. Quantitative electrophoretic mobility shift assays reveal that the specific DNA affinity of the protein is increased 10^3-fold by transfer of a methyl group to Cys; the Cd(II) and the Zn(II) forms of the protein behave similarly in this respect. This methylation-sensitive stimulation of binding underlies the ability of Ada to activate inducibly the transcription of a methylation-dependent regulon. We conclude that the chemical properties of the bound metal influence the transition state for autocatalytic methyl transfer, but not the structure that ultimately results from this process.


INTRODUCTION

The Ada protein directs the DNA repair response of Escherichia coli to damage by alkylating agents(1) . Ada itself participates in the repair of a mutagenic lesion, O^6-methylguanine, by direct, irreversible transfer to a cysteine residue in the C-terminal domain(2, 3) . Ada also repairs the S(p) diastereomer of DNA methylphosphotriesters (MePs) (^1)by direct transfer to another cysteine residue, Cys, located in the N-terminal domain(4) . In vitro experiments have shown that methylation of N-Ada enables the protein to activate transcription of a methylation resistance regulon and thereby direct the adaptive response to alkylating agents(5) . The known genes under the Ada regulon comprise the ada gene itself as well as several others that encode DNA repair functions. DNaseI footprinting data have shown that the methylation-dependent increase in Ada's transcriptional activation function correlates directly with an increase in specific affinity for a sequence element located directly upstream of the RNA polymerase binding site in the promoters of Ada responsive genes(5, 6, 7) .

The process of methylphosphotriester repair by the N terminus of Ada, which triggers the protein's ability to function as a transcriptional activator, has been elucidated through a combination of structural and biochemical studies on the protein. The N-terminal domain of Ada possesses a tightly bound Zn(II) ion that is necessary for proper folding in vitro and in vivo(8) . Cd nuclear magnetic resonance (NMR) studies on the N terminus of Ada (9) and a NMR solution structure of N-Ada10(10) , a 10-kDa fragment of the N terminus of Ada that retains zinc binding and DNA MeP repair functionality (Fig. 1), reveal an unusual Cys-X^3-Cys-X-Cys-X^2-Cys ligand arrangement about the metal. These structural studies identify the N terminus active site cysteine, Cys, as one of the four metal ligand residues in Ada, thereby implicating the metal ion not only in stabilization of the protein structure, but also in direct metalloactivation of the methyl acceptor residue.


Figure 1: Functionality of Ada fragments. A, schematic representation of Ada and its fragments. Positions of the Zn(II) ligand residues within the N-terminal domain are denoted above the diagram in one-letter code. The active site cysteine, Cys, is denoted by an asterisk (*). B, the Ada box. The synthetic binding site, from the ada promoter, used for gel retention assays. The 8-base sequence referred to as the Ada box is shown in boldface type. C, summary of N-Ada's repair and inducible DNA binding functionalities. The 20-kDa N-terminal domain (N-Ada20) repairs the S(p) diastereomer of methylphosphotriesters and, in the methylated form, binds DNA sequence specifically. This domain retains its activity as an individual unit. All the functionality in N-Ada20 is retained in the first 153 residues of the N terminus, N-Ada17, while only the methylphosphotriester repair and zinc binding functionality are retained in the first 92 residues of the N-terminal, N-Ada10. The metalloactivation of the methyl acceptor residue, Cys, is shown schematically. S refers to cysteine sulfhydryl groups of particular residues.



In the present study, kinetics measurements and DNA binding assays on various forms of Ada were pursued to further study the effect of the bound metal on methylphosphotriester repair and sequence-specific DNA binding by the methylated N terminus of Ada (S-Me-CysN-Ada). The execution of these studies as well as future structural studies of the methylated form of the protein required the development of a method for specific methylation of Ada, which was accomplished by means of a singly methylated oligonucleotide. Using this synthetic substrate, it was found that although the type of metal bound by Ada altered the kinetics of DNA methylphosphotriester repair, it had little effect on the sequence-specific DNA binding properties of the S-Me-CysN-Ada. Quantitative electrophoretic mobility shift assays reveal that the difference in free energy of specific DNA binding by N-Ada and S-Me-CysN-Ada is approx 4.2 kcal/mol at 25 °C.


MATERIALS AND METHODS

Synthesis of T(OMe)

The synthesis of T(OMe) was performed on an Applied Biosystems 380A DNA synthesizer using a standard coupling cycle. To a CPG-thymidine resin (Milligen), 4 units of 4,4`-dimethoxytrityl-thymidine beta-cyanoethyl phosphoramidite, 1 unit of 4,4`-dimethoxytrityl-thymidine methylphosphoramidite (Sigma), then 5 more units of 4,4`-dimethoxytrityl chloride-thymidine beta-cyanoethyl phosphoramidite were coupled followed by a final detritylation step. Deprotection of T(OEtCN)(9)(OMe) (Fig. 2) followed the procedure of Kuijpers et al.(11) , with some modifications. For a 0.2-µmol synthesis, deprotection was performed with 150 µl of 0.05 M K(2)CO(3) in anhydrous methanol at room temperature for 7 h. The solution was neutralized by adding Dowex H resin (prewashed with anhydrous methanol) a few beads at a time, then checking the pH using indicator paper. H(2)O (200 µl) was added, the suspension was centrifuged, and the supernatant was removed. The resin was then washed with another 200 µl of H(2)O. The combined supernatants were evaporated and resuspended in 200 µl of TE (10 mM Tris (pH 7.0), 1 mM EDTA). These values were scaled five times for a 1.0 µmol synthesis. Control oligonucleotides were synthesized by the following procedures: (i) treatment of T(OEtCN)(9)(OMe) with concentrated NH(4)OH, 55 °C, 16 h; (ii) synthesis of an 11-mer using only 4,4`-dimethoxytrityl-thymidine beta-cyanoethyl phosphoramidite (T(OEtCN)), followed by deprotection using (a) K(2)CO(3)/MeOH as described above or (b) concentrated NH(4)OH, 55 °C, 16 h. Oligonucleotides were quantified by UV and used without further purification.


Figure 2: Scheme for T(OMe) synthesis and repair. The synthetic scheme for the T(OMe) substrate and the observed yields for the products are summarized above.



FPLC Analysis

Oligonucleotides (100-1000 pmol) were dissolved in buffer A (20 mM Tris, pH 8.1), passed through a 0.22-µm microcentrifugal filtration unit (Vanguard, Neptune, NJ), and loaded on a Pharmacia Mono Q HR 5/5 column equilibrated with 87% buffer A, 13% buffer B (buffer A plus 1.5 mM NaCl), at a flow rate of 0.5 ml/min. After eluting 4 ml at 13% buffer B, a 14-ml gradient was run to 28% buffer B, followed by a 1-ml gradient to 100% buffer B, a 4-ml isocratic elution at 100% B, and then a 4-ml gradient to 13% buffer B. In this system, T(OMe) eluted at 300 mM NaCl and T at 330 mM (see below).

Protein Preparations

The 17-kDa N-terminal domain of Ada (N-Ada17) was overproduced by a polymerase chain reaction-based approach using the 5`-primer for the promoter region of the vector pN-Ada20, in combination with a 3`-primer that encoded a stop codon and a HindIII restriction site following Ser. The polymerase chain reaction product was digested with EcoRI and HindIII, ligated into pKEN1(12) , and transformed into XA 90 to yield the overproducing phagemid pN-Ada17.

The N-Ada17 protein was purified according to the same procedure used for N-Ada20(8) . The other zinc-bound forms of Ada and its N-terminal fragments were purified as described(8) . Cadmium-bound forms of these proteins were generated by growth in metal-supplemented minimal media as described previously(9) .

Protein concentrations were measured by the Bradford assay, using as calibration standards Ada samples that had been independently quantified by amino acid analysis.

Phosphotriester Repair Assay

Typically, between 200 and 600 pmol of protein was incubated with T(OMe) in 6-50 µl of assay buffer (50 mM Hepes-KOH (pH 7.8), 10 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 50 µM spermidine hydrochloride) at different temperatures for various amounts of time. Control reactions in which the protein was omitted were also performed. The stoichiometry of the reaction was determined by either incubating varying amounts of protein (0.1, 0.5, 0.8, 1, 2, 4, and 10 eq) with a constant amount of DNA (200 pmol of total DNA) or by incubating a constant amount of protein (150 pmol) with varying amounts of T(OMe) (200, 330, 550, and 720 pmol of total DNA), at 4 °C or 25 °C for greater than 1 h. Overnight incubations were also carried out to ensure that the reactions had proceeded to completion. Reactions were worked up by the addition of Buffer A to 200 µl, filtration, and FPLC analyses as described above. Values were corrected for the percentage of S(p)-T(OMe) in the starting DNA (typically 40% of total DNA) as determined by P NMR (13) .

Kinetics of Phosphotriester Repair

In order to determine bimolecular rate constants (k(2)) for phosphotriester repair by different fragments of the protein, repair reactions were performed so that the initial concentrations of active protein and S(p)-T(OMe) were equal. Under these conditions, a plot of the reciprocal of the S(p)-T(OMe) concentration versus time is linear with slope equal to k(2). Typically, 120 pmol of active protein and S(p)-T(OMe) were incubated in 21 µl of assay buffer at 4 °C. The reactions were quenched at different times by addition of 180 µl of a 7 M urea solution at 4 °C and immediately followed by FPLC analysis.

Nucleoside Composition Analysis

Oligonucleotide samples (20 nmol) were digested with 0.1 unit of snake venom phosphodiesterase and 75 units of Benzonase in 10 mM MgCl(2), 70 mM NaCl, 70 mM Tris-HCl (pH 7.5) at room temperature for 45 min, and 2 units of alkaline phosphatase in 100 mM Tris-HCl (pH 7.5), 0.1 mM ZnCl(2), 40 mM NaCl, 10 mM beta-mercaptoethanol at 37 °C for 15 min. Samples were passed through a Millex 22-µm filter, then analyzed by reverse phase high performance liquid chromatography on a Hewlett-Packard 1090 diode array system; solvent A: 0.02 M KH(2)PO(4), pH 5.6; solvent B: 60:40 MeOH/H(2)0; 1.5 ml/min; elution program: isocratic A for 1 min, 0-25% B in 10 min, 25-100% B in 5 min, isocratic B for 10 min. Authentic standards were used for comparison of UV spectrum and retention time. Nucleosides were identified by comparison with authentic standards. Blank runs in which no oligonucleotide was added to the digest served as a control for peaks that were introduced along with the protein preparations.

Gel Retention Assay

Oligonucleotides bearing the Ada binding site ``Ada box'' (Fig. 1) were produced using an Applied Biosystems 380A DNA synthesizer. A synthetic 33-mer, 5`-(d-CAGCGAAAAAAATTAAAGCGCAAGATTGTTGG)-3` (Ada box in bold), and its complement were purified by gel electrophoresis under denaturing conditions (7 M urea, 20% polyacrylamide), eluted in 1 M triethylammonium bicarbonate, and concentrated using a Sep-Pak cartridge according to the manufacturer's protocols (Waters). Equimolar amounts of the complementary purified oligonucleotides were annealed and end-labeled by the polynucleotide kinase reaction following protocols provided by the manufacturer (New England Biolabs).

For analyzing the DNA binding properties of the unmethylated protein, Ada preparations were incubated with P-labeled duplex oligonucleotides containing the Ada box for 15 min in binding buffer (50 mM TrisbulletHCl (pH 7.8), 1 mM EDTA, 5% glycerol, 10 mM dithiothreitol, 50 µg/ml bovine serum albumin) at 25 °C. For analysis of the S-Me-CysAda protein fragments with zinc incorporated, the unmethylated protein was added to a mixture of the P-labeled duplex 33-mer fragments and the T(OMe) substrate (approx10^4 molar excess over protein concentration) and incubated for 40 min at 25 °C in binding buffer. The reactions for the cadmium-containing S-Me-CysAda fragments were conducted in a similar manner except that the incubation times were extended to 90 min. For binding studies in which CH(3)I was the methyl donor, neat CH(3)I (Sigma) was diluted in dimethyl sulfoxide to a concentration of 1.6 mM, added in a 1:50 ratio to a mixture of the Ada fragments plus the P-labeled 33-mer in binding buffer, and incubated for 30 min. The total reaction volume used in each case was 50 µl. Poly(dIbulletdC) was added to some binding experiments, where indicated, to test for specificity of binding.

For all proteins tested, a total volume of 50 µl of reaction mixture was loaded on to a 4% polyacrylamide gel prepared in 40 mM TrisbulletAcOH (pH 8.2), 1 mM EDTA, and 5% glycerol. The running buffer was 40 mM TrisbulletAcOH (pH 8.2), 1 mM EDTA. After electrophoresis for about 2 h at 140 V/40 mA, the gels were dried under vacuum, exposed to a Fuji phosphoimaging plate, and analyzed using a Fuji Bio-Image analyzer.

Determination of Equilibrium Binding Constants

Binding constants were determined by the Scatchard plot method of analysis as described by Meisterernst et al.(14) . Using this method, the concentration of the protein was kept constant and the dissociation constant (K(d)) was derived from the following equation: [PbulletD]/[D] = (1/K(d)) times [P]^0 - (1/K(d)) times [PbulletD], where [D] is the concentration of unbound DNA, [P]^0 is the total protein concentration, and [PbulletD] is the concentration of the proteinbulletDNA complex. Thus, a value for K(d) can be derived from the slope of the line described above when [PbulletD]/[D] is plotted against [PbulletD].


RESULTS

Synthesis of T(OMe) DNA

To circumvent several problems with previous methods of substrate preparation and use (see ``Discussion''), we decided to synthesize a homogenous methylphosphotriester-containing DNA substrate. For reasons of synthetic simplicity, we focused our initial efforts on synthesis of a single-stranded thymidine homopolymer containing a single, centrally located methyl phosphotriester; this molecule is designated T(OMe) (Fig. 2). Among the objectives of the present study was to determine whether this single-stranded molecule would function as an efficient Ada substrate.

T(OMe) was synthesized according to the method described by Kuijpers et al.(11) , as illustrated in Fig. 2. A resin-bound 11-mer with nine internucleotide beta-cyanoethyl phosphotriester linkages and one methyl phosphotriester linkage, T(OEtCN)(9)(OMe), was treated with K(2)CO(3) in MeOH, which was expected to cleave the beta-cyanoethyl protecting groups and the 3`-linkage to the support, yet leave the methyl phosphotriester largely intact. The reaction products were then analyzed by anion exchange FPLC. As shown in Fig. 3A, two products were obtained from the reaction, a major component eluting at 10 ml and a minor one at 12 ml. The 12-ml product was identified as the fully deprotected 11-mer, T, since it co-eluted on FPLC with an authentic standard synthesized by conventional methods (Fig. 3C). The 10-ml peak was identified as T(OMe) by nucleoside composition analysis, which yielded dT and T(2)(OMe) (data not shown). Treatment of T(OEtCN)(9)(OMe) with concentrated aqueous ammonia yielded only the 12-ml peak (data not shown), consistent with the known hydrolytic lability of methyl phosphotriesters(15) . The ability of anion exchange FPLC to distinguish between T(OMe) and T provided a convenient means to determine both the amount of phosphotriester hydrolysis that accompanies deprotection of T(OEtCN)(9)(OMe), as well as the rate and extent of methyl transfer to Ada (see below). In the several syntheses we have carried out, the ratios of T(OMe) to T typically range from 75%:25% (10 µmol scale) to 90%:10% (0.2 µmol scale). By this route, it is regularly possible to synthesize milligram quantities of Ada substrate.


Figure 3: FPLC analysis of T(OMe) synthesis and repair. A, the elution profile of 200 pmol of untreated T(OMe), as synthesized by the scheme outlined above, using anion exchange chromatography. The two peaks labeled are identified via nucleoside composition analysis as described above. B, the result of treatment of 200 pmol of the T(OMe) substrate with an equimolar amount of N-Ada20. The remaining T(OMe) substrate is presumably the unrepaired R(p) diastereomer. C, the elution profile of 200 pmol of untreated T synthesized by standard methods.



Although for the present studies there was no need to separate T(OMe) from T, these should be easily separable by anion exchange chromatography, as suggested by the trace in Fig. 3A. In our hands, however, we have not been able to separate chromatographically the two diastereomeric components of T(OMe), namely S(p)-T(OMe) and R(p)-T(OMe) (refer to Fig. 2).

Demonstration of T(OMe) as Substrate of Ada in the Repair of Phosphotriesters

Incubation of T(OMe) with the 20-kDa N-terminal fragment of Ada (N-Ada20) was found to decrease the ratio of T to T(OMe) relative to the starting DNA (compare Fig. 3B with 3A). When limiting amounts of protein were added, the conversion from T(OMe) was proportional to the protein concentration (1:1 ratio). Changing neither the incubation time from 5 min to 12 h nor the reaction temperature from 22° to 37 °C had a significant effect in the product conversion, indicating that the reaction goes to completion within 5 min. Thus, even a single-stranded 11-mer is an efficient substrate for the phosphotriester repair domain of Ada.

Methyl transfer reactions using excess protein over T(OMe) invariably reached a plateau when approx55% of the starting T(OMe) had been converted to T. This observation is consistent with the known stereospecificity of the methyl transfer reaction(16) ; namely, Ada repairs only the S(p) diastereomer and leaves the R(p) diastereomer unrepaired(13) . Since no specific measures were taken to impose stereoselectivity upon the synthesis of the methylphosphotriester in T(OMe), it is to be expected that the two isomers are formed in roughly equal proportions. In contrast to the efficient repair of T(OMe), Ada was found not to demethylate a phosphotriester-containing dimer, T(2)(OMe).

Although no attempt was made to analyze the protein product of the reactions with T(OMe), data discussed below reveals that methylation of Ada by T(OMe) potentiates sequence-specific binding, consistent with the notion that T(OMe) transfers its methyl group to Cys in much the same fashion as in vitro-methylated duplex DNA.

Methyl Transfer Kinetics of Ada and Ada Fragments

To determine the relationship of Ada domain organization and the rate of phosphotriester repair, we examined a series of Ada protein species: full-length (39-kDa) Ada, the entire N-terminal domain as defined by proteolytic processing (N-Ada20)(4, 8) , and an N-terminal fragment containing the intact zinc-binding module of Ada (residues 1-92, N-Ada10), but lacking residues that are necessary for sequence-specific DNA binding(17) . These various forms of Ada have been expressed at high levels in E. coli and purified to apparent homogeneity; the solution structure of N-Ada10 has been reported(10) . Furthermore, by an appropriate choice of growth conditions it is possible biosynthetically to charge the Ada fragments with either Zn or Cd(9) . As mentioned above, demethylation of T(OMe) by the zinc-bound form of N-Ada20 (N-Ada20[Zn]) is complete within 5 min at 25 °C; under these conditions, the reaction is too fast to obtain kinetics measurements without employing stopped flow techniques. However, by lowering the reaction temperature to 4 °C, it was possible to slow the reaction sufficiently that kinetics measurements could be obtained in a straightforward manner. Through titration of the Ada fragments with T(OMe), the activity of each protein is first determined, then used to adjust the protein concentration in kinetics measurements such that the T(OMe) and active protein were present at unit stoichiometry. As controls, we tested for product inhibition of the methyl transfer by T and for DNA demethylation by thiols present in the protein preparation. End product inhibition was not observed, even when T was present at 1000-fold excess over T(OMe); furthermore, concentrations of glutathione 10^5 times above that of Ada led to no detectable loss of T(OMe) (data not shown). The kinetics of phosphotriester repair by the various Ada forms was determined by mixing together the protein and T(OMe), periodically removing aliquots and quenching with urea, then analyzing the extent of conversion of T(OMe) to T using the FPLC assay. The bimolecular rate constant, k(2), is derived from plots of time versus the reciprocal of the T(OMe) concentration (Fig. 4, Table 1). Not only were N-Ada20[Zn] and N-Ada10[Zn] fully active in phosphotriester repair, but they repair the lesion more rapidly than Ada itself (Table 1).


Figure 4: Methylphosphotriester repair rate measurements for various forms of Ada. To determine bimolecular rate constants (k(2)) for phosphotriester repair by different fragments of the protein, repair reactions were performed so that the initial concentrations of active protein and S(p)-T(OMe) were equal. Under these conditions, a plot of the reciprocal of the S(p)-T(OMe) concentration versus time is linear with slope equal to k(2).





Structural and biochemical studies have shown that the methyl acceptor residue, Cys, is coordinated to the metal ion in Ada. Although the zinc- and cadmium-bound forms of the N-terminal domain are very similar in structure, the two forms are likely to differ in the electronic properties of Cys, which may manifest itself in rate differences. Indeed, the cadmium-bound forms of N-Ada10 and N-Ada20 repair methylphosphotriesters roughly 4-fold more slowly than the corresponding zinc-bound counterparts. These findings are consistent with the notion that the metal is involved in the rate-determining step for methyl transfer(9) .

DNA Binding Characteristics of Ada

Following methylation of Cys, the N-terminal domain of Ada acquires the ability to bind specifically to DNA sequences in the ada and alkA promoters(7) . To quantify the magnitude of the methylation-dependent effect on the DNA-affinity of Ada, and the effects of truncation and metal binding, we carried out a series of electrophoretic mobility shift assay experiments (Fig. 5). In preliminary experiments, the ability to obtain accurate binding data was found to be dramatically affected by the conditions under which the methylation, protein-DNA incubation, and electrophoresis took place. In particular, high ionic strength buffers for the methyl transfer reaction and subsequent electrophoresis were necessary to obtain sharp bands for the protein-DNA co-complexes found with both forms of the proteins. Several additional measures were necessary to generate and maintain full activity of the methylated protein. Methylation of the N terminus is accompanied by a substantial increase in the lability of the protein toward denaturation; this can be corrected by methylating the protein in the presence of a double-stranded oligonucleotide containing a specific recognition site, the so-called Ada box. The presence of the Ada box oligonucleotide, however, was observed to impede the methyl transfer reaction, presumably due to preferential binding of the unmethylated N-terminal domain to the duplex DNA. Titrations of the protein with increasing amounts of TOMe (data not shown) revealed that methylation was most efficient when an approx10^4 molar excess of TOMe over protein was used over a 30-min incubation. On the other hand, only when the single-stranded DNA was present in 10^7-fold excess over duplex was competition against sequence-specific recognition observed. Lastly, to achieve full activity of the oxidation-sensitive Ada protein, it was found that it is crucial to use freshly prepared solutions of dithiothreitol in the reaction buffer. With these conditions worked out, we were able to obtain quantitative data on the sequence-specific interaction of Ada with the Ada box.


Figure 5: Gel electrophoresis and Scatchard plot of zinc S-Me-CysN-Ada17-Ada box interactions. A, electrophoretic mobility shift assay on zinc S-Me-CysN-Ada17. All data shown above the gel are picomolar concentrations. Lane 1 contains only the free oligonucleotide probe. A saturation of the binding site with excess protein in lane 2 demonstrates that the synthetic probe is approx80% active. This factor is taken into account in all further quantitation of bound and free probe. Lanes 3-6 show the titration of a constant amount of protein with an increasing concentration of labeled probe. B, the data were obtained from gel retention assays (shown above) and analyzed according to Equation 1. [D] is the experimentally determined concentration of the unbound Ada binding site, and [PbulletD] is the experimentally determined concentration of the Ada binding site complexed with the protein.



To facilitate future structural studies of the protein, we have attempted to truncate Ada to the minimal fragment capable of methylation-dependent DNA recognition. The smallest Ada fragment known to retain phosphotriester repair activity, N-Ada10, lacks sequence-specific DNA binding(17) . Taking into account sequence homology among Ada proteins(18) , secondary structure predictions, and limited proteolysis data(4) , we had reason to believe that an Ada fragment comprising residues 1-153 (N-Ada 17) would possess both phosphotriester repair and sequence-specific DNA binding affinities. Electrophoretic mobility shift assay analysis of S-Me-CysN-Ada17[Zn] alongside S-Me-CysAda[Zn] reveals that the truncated protein binds DNA with an affinity indistinguishable from that of its full-length parent (Table 2). Binding of unmethylated N-Ada17 also exhibited excellent selectivity, being unaffected by 1000-fold excess of nonspecific competitor DNA (poly(dIbulletdC)) (data not shown). The picomolar DNA affinity of S-Me-CysAda[Zn] and S-Me-CysN-Ada17[Zn] rivals that of any known protein that, like Ada, binds DNA as a monomer (see (19) for summary of DNA-protein binding constants).



Footprinting studies (7) and in vitro transcription assays (6) have demonstrated that methylation of Cys in Ada enables the protein to activate transcription by enhancing its specific affinity for Ada box sequences in the ada and alkA promoters. The magnitude of this enhancement, however, has not been reported. Overexpression of Ada results in activation of the ada promoters, suggesting that even the unmethylated protein possesses some specific affinity for DNA. To address this issue, we measured the specific affinities of N-Ada17[Zn] for the Ada box. Indeed, N-Ada17[Zn] was found to bind the Ada box specifically, with a K(d) of 1.2 ± 0.4 times 10M. Thus, methylation of Cys in Ada stimulates binding to the Ada box approx10^3-fold. Interestingly, the residual DNA affinity of N-Ada17 shows pronounced metal-ion dependence: N-Ada17[Cd] was found not to bind the Ada box specifically.

Evidence has been presented that some weak methylating agents, such as CH(3)I, can induce the adaptive response by direct alkylation of Ada. To assess the effect of CH(3)I on the sequence-specific binding function of Ada on the sequence-specific binding function of Ada, we pretreated N-Ada17[Zn] with CH(3)I, in the presence of DNA, and then measured the affinity of the protein for the Ada box site. Indeed, N-Ada17[Zn], treated with CH(3)I, bound DNA just as tightly as the T(OMe)-treated protein (Table 2). Thus, simple alkylating agents such as CH(3)I are able to produce the same activation stimulus as that ordinarily brought about by DNA phosphotriester repair.

In another series of binding experiments, we measured the effect of the metal in DNA binding by methylated N-Ada17. Consistent with the above kinetics data described above, the incubation of the cadmium protein with T(OMe) took 60 to 90 min longer to reach full binding activity than necessary for the zinc protein. Just as the cadmium form of Ada is slightly impaired in methylation kinetics, upon methylation, the cadmium protein also exhibits moderately reduced (approx10-fold) affinity for DNA. Nonetheless, S-Me-CysN-Ada17[Cd] binds DNA with exceptionally high strength and specificity, and thus this form of the protein appears suitable for structural studies aimed at elucidating the molecular basis of methylation-dependent structural switching in Ada.


DISCUSSION

In the experiments reported herein, we have found that a single-stranded homopolymer containing a single methyl phosphotriester, T(OMe), efficiently and quantitatively methylates the N terminus of Ada. These findings are in contrast to a report claiming that single-stranded DNA is a poor substrate for phosphotriester repair by N-Ada(20) . Access to a homogeneous oligonucleotide substrate for Ada provided the basis for a convenient method to assay its activity. In previous studies of phosphotriester repair by Ada, the repair substrate was commonly generated by treating poly(dT) with methylating agents, typically N-methyl-N-nitrosourea (MNU) or N-methyl-N`-nitro-N-nitrosoguanidine (MNNG), and subsequently annealing with poly(dA) to form the double-stranded substrate(16) . Although this approach was convenient for small-scale methylation of Ada, particularly using radiolabeled substrate, it also presents several problems: (i) such methylated DNA preparations can contain some proportion of a substrate for the C-terminal domain of Ada, O^4-methylthymine, in addition to the methylphosphotriester substrate for the N-terminal domain, thus making it difficult to alkylate selectively the N-terminal domain; (ii) the structural nonhomogeneity of the substrate presented problems for acquisition and interpretation of kinetics data; (iii) the synthesis requires handling of toxic and carcinogenic materials, which could be especially problematic with large-scale preparations; and (iv) given the extremely poor efficiency of the DNA methylation reactions, it would be very difficult to generate sufficient quantities of substrate to support structural and mechanistic studies. The T(OMe) substrate avoids these shortcomings. Our studies suggest that MeI may be an even simpler alternative for large-scale methylation of N-Ada; however it remains to be shown that MeI is specific for only Cys.

Kinetics studies, using the T(OMe) substrate, have helped elucidate the role of the bound metal in methyl transfer to N-Ada. Structural studies have identified the active site cysteine, Cys, as a metal ligand in the unmethylated form of N-Ada (9, 10) . The decrease in rate of MeP repair from the zinc form to the cadmium form indicates that the physicochemical properties of the metal are involved in the reaction mechanism. Cadmium and zinc both possess the same valence shell electronic configuration (d) and thus exhibit similar preferences with respect to ligand atom type and arrangement. Akin to many other metalloproteins, such as GAL4(21) , the cadmium form of N-Ada10 is close in secondary and tertiary structure to that of the native zinc form(9) . The atomic radius and the thiol-metal bond length of Cd(II), however, are both larger than that for Zn(II)(22) .

The differences in MeP repair activity for the Zn(II) and Cd(II) form of Ada can be interpreted within the large body of data characterizing the zinc enzyme carbonic anhydrase. Earlier, it was noted that the role of the metal in Ada could be viewed analogously to the role played by zinc in the activation of H(2)O by carbonic anhydrase(9) . Namely, as the bound zinc in carbonic anhydrase lowers the pK(a) of the coordinated solvent(23) , the metal in Ada effectively lowers the pK(a) of the coordinated cysteine thiol. Thus, the metal assists in the deprotonation of the protonucleophile, presenting a metal-bound anion for reaction with an electrophile. Esterase activity measurements for the zinc and cadmium forms (24) and the manganese form (25) of carbonic anhydrase show a correspondence between the pK for the activity and the ionic radius of the metal ion. As the ionic radius increases, the bound metal in carbonic anhydrase becomes much less effective in lowering the pK(a) of the coordinated solvent. Studies on pK values for a hydrolytic water molecule in different metal complexes showed a similar correlation between metal bond length and pK values(26) . Within the scheme proposed above for the metalloactivation of the cysteine thiolate, both of these trends are consistent with the observed decrease in Ada MeP repair rate going from the Zn(II) bound to the Cd(II) form of the protein. If the kinetically competent nucleophile is a metal-bound thiolate, then the electron-accepting properties of the metal, in addition to its effects on solvation of the thiolate, may also play a role in determining the rate of reaction. Besides its role in autocatalysis, the metal in Ada also stabilizes the tertiary structure of the protein(8) , another common function for zinc in metalloproteins.

In contrast to the metal dependence of Ada's MeP repair function, the methylated Cd(II) form of the protein completely retains the sequence-specific DNA binding activity observed for the Zn(II) form. Similar data have been observed for several other proteins that utilize a native zinc to stabilize their sequence-specific DNA binding domains, such as the Cd(II)-glucocorticoid receptor (27) and Cd(II)-GAL4(28) . Whereas changing the metal has essentially no effect on the methylated N terminus, it does affect the residual DNA affinity of the unmethylated protein. The origins of this difference are unclear, given the limited information presently available on sequence-specific binding on any form of Ada. Surprisingly, we detected a small percentage (approx0.1%) of the protein that appeared to be active for binding even without exposure to exogenous methylating agents. This active component exhibited a K(d) similar to that of bona fide methylated N-Ada17[Zn], suggesting it might arise from methylation by endogenous agents, such as Sadenosylmethionine, in vivo. It is possible that other modifications of the protein, such as oxidation, could lead to gratuitous activation.

Quantitative information on the transition from the unmethylated to methylated form of Ada now enables further interpretation of published data on the conformational switch in Ada. From in vitro experiments, Teo et al.(5) estimated that the methylated Ada protein is about 100 times more efficient as a transcriptional activator than the unmethylated protein. The approx10^3-fold observed increase in sequence-specific binding at the Ada box is consistent with a scheme in which Ada's acquisition of transcriptional activator functionality is fully attributable to an increase in DNA binding affinity. Recent studies have shown that the S-methylcysteine at position 69 is coordinated to the metal of Ada when the protein is bound specifically to DNA (29) and also in the methylated protein not bound to DNA(30) . This observation seems to rule out a scenario in which methylation brings about a dramatic change in the structure of the protein. Given the information presently available, the simplest model for transcriptional switching in Ada is one in which acquisition of a methyl group on Cys introduces new functionality directly into the protein-DNA interface. Consistent with this, NMR measurements have shown that protons of the Cys methyl group experience an approx0.5 ppm downfield shift upon binding to DNA(29, 30) , suggesting the methyl group might contribute directly to the protein-DNA interface. It has been suggested that a helix-turn-helix motif located approx40 residues C-terminal to Cys is directly involved in DNA binding(31) . If so, the DNA binding domain of Ada is apparently bipartite. The difference in K(d) of 10^3 translates to a free energy difference of 4.2 kcal/mol at 25 °C. This is larger than the free energy change typically observed, for example, upon removal of a methyl group from a protein-DNA interface(32) . However, such cases generally involve simple removal of favorable contact functionality; in the case of Ada, it may be that methylation brings about both a gain in energetically favorable interactions with the methyl group and a loss of repulsive interactions experienced with the native protein. Alternatively, it is possible that minor structural adjustments brought about by methylation give rise to a more snug fit of the protein-DNA interface.


FOOTNOTES

*
This work was supported by grants from the Chicago Community Trust (Searle Scholars Program) and the National Science Foundation (Presidential Young Investigator Award Program) (to G. L. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Howard Hughes Medical Institute Predoctoral Fellow.

To whom correspondence and reprint requests should be addressed.

(^1)
The abbreviations used are: MeP, methylphosphotriester; N-Ada10, Ada fragment containing residues 1-92; N-Ada20, Ada fragment containing residues 1-178; N-Ada17, Ada fragment containing residues 1-153; S-Me-CysN-Ada, Cys-methylated N terminus of Ada; FPLC, fast protein liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Lin Chen and Christopher Larson for binding constant related discussions and Prof. G. Wagner for Ada related discussions.


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