(Received for publication, July 27, 1994; and in revised form, January 5, 1995)
From the
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
-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.
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-methylguanine, by direct, irreversible transfer
to a cysteine residue in the C-terminal domain(2, 3) .
Ada also repairs the S
diastereomer of DNA
methylphosphotriesters (MePs) (
)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
-Cys-X
-Cys-X
-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
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-Cys
N-Ada. Quantitative electrophoretic
mobility shift assays reveal that the difference in free energy of
specific DNA binding by N-Ada and S-Me-Cys
N-Ada
is
4.2 kcal/mol at 25 °C.
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.
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.
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 Tris
HCl (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-Cys
Ada 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 (
10
molar excess over
protein concentration) and incubated for 40 min at 25 °C in binding
buffer. The reactions for the cadmium-containing S-Me-Cys
Ada fragments were conducted in a similar
manner except that the incubation times were extended to 90 min. For
binding studies in which CH
I was the methyl donor, neat
CH
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(dI
dC) 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 TrisAcOH (pH 8.2), 1 mM EDTA, and 5% glycerol. The
running buffer was 40 mM Tris
AcOH (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.
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
-cyanoethyl phosphotriester linkages and one methyl
phosphotriester linkage, T
(OEtCN)
(OMe), was
treated with K
CO
in MeOH, which was expected to
cleave the
-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
(OMe) (data not
shown). Treatment of T
(OEtCN)
(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)
(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
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
-T
(OMe)
and R
-T
(OMe) (refer to Fig. 2).
Methyl transfer reactions
using excess protein over T(OMe) invariably reached a
plateau when
55% 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
diastereomer and
leaves the R
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
(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.
Figure 4:
Methylphosphotriester repair rate
measurements for various forms of Ada. To determine bimolecular rate
constants (k) for phosphotriester repair by
different fragments of the protein, repair reactions were performed so
that the initial concentrations of active protein and S
-T
(OMe) were equal. Under these
conditions, a plot of the reciprocal of the S
-T
(OMe) concentration versus time is linear with slope equal to k
.
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) .
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-Cys
N-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
80% 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 [P
D] 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-Cys
Ada[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(dI
dC)) (data
not shown). The picomolar DNA affinity of S-Me-Cys
Ada[Zn] and S-Me-Cys
N-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
of 1.2 ± 0.4
10
M. Thus, methylation of
Cys
in Ada stimulates binding to the Ada box
10
-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 CHI,
can induce the adaptive response by direct alkylation of Ada. To assess
the effect of CH
I on the sequence-specific binding function
of Ada on the sequence-specific binding function of Ada, we pretreated
N-Ada17[Zn] with CH
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
I, bound DNA just as
tightly as the T
(OMe)-treated protein (Table 2).
Thus, simple alkylating agents such as CH
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 (
10-fold) affinity for DNA.
Nonetheless, S-Me-Cys
N-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.
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
-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 HO by carbonic anhydrase(9) .
Namely, as the bound zinc in carbonic anhydrase lowers the
pK
of the coordinated solvent(23) , the
metal in Ada effectively lowers the pK
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
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 (0.1%) of the protein that appeared to be active
for binding even without exposure to exogenous methylating agents. This
active component exhibited a K
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 10
-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
0.5 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
40 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
of 10
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.