Download Active site amino acid sequence of the bovine O6

Nucleic Acids Research, Vol. 18, No. 1
© 1990 Oxford University Press
Active site amino acid sequence of the bovine
O 6-methylguanine-DNA methyltransferase
Bjorn Rydberg, Janet Hall* and Peter Karran
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK
Received October 2, 1989; Revised and Accepted November 10, 1989
ABSTRACT
An O6-methylguanine-DNA methyltransferase has
been partially purified from calf thymus by conventional
biochemical techniques. The enzyme was specifically
radioactively labelled at the cysteine residue of the
active site and further purified by attachment to a solid
support. Following digestion with trypsin, a radioactive
peptide containing the active site region of the protein
was purified by size fractionation, ion exchange
chromatography and reverse phase HPLC. The
technique yielded an essentially homogeneous
oligopeptide which was subjected to amino acid
sequencing. The sequence adjacent to the acceptor
cysteine residue of the bovine protein exhibits striking
homology to the C-terminal methyl acceptor site of the
E. coli Ada protein and the proposed acceptor sites of
the E. coli Ogt and the B. subtilis Dat1 proteins.
INTRODUCTION
O6-Methylguanine (m6-Gua) is one of the major products of the
reaction of a number of methylating agents with DNA and is
responsible for the potent mutagenicity of carcinogens such as
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the
metabolically activated form of dimethylnitrosamine (1). The
spectrum of mutations induced by this group of methylating agents
is dominated by GC to A T transition mutations (2,3) which is
consistent with the observed propensity of m6-Gua to direct the
incorporation of thymine into DNA in vitro (4). In addition to
its well-documented mutagenicity, m6-Gua also contributes to
other biological effects of alkylating agents. A number of human
and rodent cell lines are unable to remove m6-Gua from their
DNA. These cell lines are designated Mex~ (or Mer~) and are
hypersensitive to the cytotoxic, clastogenic and mutagenic action
of methylating agents (5,6). The hypersensitivity of cells which
do not remove m6-Gua can be completely reversed by
expression of a transfected E. coli ada+ gene encoding an
m6-Gua repair function (7-9). Thus, m6-Gua is strongly
implicated not only in the mutagenic, but also in the cytotoxic
and clastogenic action of agents such as MNNG towards
mammalian cells.
In many bacteria (including E. coli (10), M. luteus (11), and
B. subtilis (12)) the first line of defence against the biological
effects of this methylated base is repair by specific DNA
methyltransferases which demethylate the modified purine in situ
but are without effect on other chemically-induced or naturally
occurring methylated bases. The bacterial methyltransferases
include the inducible Ada protein, a dual function
methyltransferase comprising two domains which act separately
to demethylate m6-Gua or methylphosphotriesters (10), and the
constitutively expressed Ogt proteins off. coli (13) and the Datl
(14) protein of B. subtilis. A common feature of these bacterial
methyltransferases is transfer of a methyl group from the modified
purine base onto a particular receptor cysteine residue within the
methyltransferase molecule itself; the transfer being accompanied
by an irreversible inactivation of the methyltransferase function.
m6-Gua-DNA methyltransferase activities have been partially
purified from several mammalian sources (15-18) and
preliminary characterisation has indicated that they share a
number of features with their bacterial counterparts. In particular,
the automethyltransfer mode of repair has apparently been
conserved and mammalian cells from a variety of sources
(including human) are able to demethylate m6-Gua both in vivo
and in cell-free extracts. In all cases, removal of methyl groups
from m6-Gua in DNA is accompanied by a stoichiometric
production of S-methylcysteine in a protease-sensitive form
indicating that a cysteine residue serves as acceptor.
Despite considerable efforts in a number of laboratories,
mammalian methyltransferases have proved refractory to high
yield purification and this has hampered further clarification of
the mechanism of action of this important DNA repair enzyme.
These difficulties have been partly due to excessive losses of the
partially purified enzyme during the final stages of purification.
Here we report the isolation and amino acid sequence of a peptide
comprising the active site of the bovine enzyme. The derived
sequence demonstrates a remarkable homology to the m6-GuaDNA methyltransferase active site of the E. coli Ada protein and
the putative active site sequences of the E. coli Ogt and the B.
subtilis Datl proteins.
MATERIALS AND METHODS
Materials
Trypsin (Sequencing Grade) was obtained from Boehringer
Mannheim. Sephadex G25 Superfine and the MonoS FPLC
cation exchange column were obtained from Pharmacia and
Ultrogel AcA54 from LKB. DE52 ion exchange cellulose and
* Present address: International Agency for Research on Cancer, 150 Cours Albert Thomas, 69372 Lyon, France
17
18 Nucleic Acids Research
phosphocellulose PI 1 (Whatman) were prepared according to the
manufacturers instructions. Single-stranded DNA cellulose was
obtained from Sigma.
Synthetic Oligonucleotides
The 21mer oligonucleotide 5'-TGATCAGTAC(m6-G)CATGACTAGT-3' was synthesised by the phosphoramidite method on
an Applied Biosystems Model 380B DNA synthesiser and further
purified by HPLC. For use as a substrate for the
methyltransferase, it was annealed to the oligomer 5'-ACTAGTCATGCGTACTGATCA-3' at a concentration of 2/iM in 0.1 M
NaCl, lOmM TrisHCl pH 7.5, lmM EDTA at 37°C for
60min.
m'-Gua-DNA Methyltransferase Assay
[3H]-methylated M. luteus DNA was prepared using [3H]-Nmethyl-N-nitrosourea (MNU) (Amersham International,
24Ci/mmole) and partially depurinated as described (19). Assays
were carried out in: 70mM Hepes KOH, pH 7.8/ lOmM
dithiothreitol/ lmM EDTA. To monitor the purification
procedure, 0.1— 2/il of each column fraction was incubated in
100/il reaction buffer containing [3H] substrate (lOOOcpm) for
20min at 37 °C. Following a digestion with proteinase K
(125^g/120min) at 37°C, nucleic acids were precipitated with
ethanol and the [3H] radioactivity in the supernatant was
determined by scintillation counting. When appropriate, the
removal of m6-Gua from the DNA was also monitored by
published procedures (18).
Partial Purification of m6-Gua-DNA Methyltransferase from
Calf Thymus
The procedure is based on that reported by Hall and Karran (18).
All operations were performed at 0 - 4 ° C . 1.8Kg calf thymus
freshly obtained from the slaughterhouse, was homogenised in
4 1 extraction buffer (0.1M NaCl, 50mM TrisHCl pH 7.5,
lmM EDTA, 0.1% /3-mercaptoethanol, 0.1% Triton X-100,
lmM phenylmethylsulphonyl fluoride, and 0.5/ig/ml each:
leupeptin, pepstatin and chymostatin) using a Waring blendor at
maximum setting for 2 x30sec. Extraction was for 45min at 0°C.
Tissue debris was then removed by centrifugation at 3200 Xg
for 30 min. To 41 supernatant was added a thick slurry of 1.21
DE52 in buffer A (50mM NaCl, 20mM Tris HC1 pH 7.5, lmM
K 2 HPO 4 , lmM EDTA, lmM dithiothreitol, 0.1% |3mercaptoethanol). The mixture was stirred for 30min and the
DE52 allowed to settle. The supernatant was decanted and the
DE52 was then washed with 41 buffer A. To the two combined
supernatants (81) was added 21 phosphocellulose PI 1 as a thick
slurry in buffer A. The mixture was stirred for lhr, the PI 1 was
then allowed to settle and the supernatant was discarded. The
PI 1 was washed twice with 81 buffer A and then poured into
a column (35cmx8.5cm diam.) which was eluted with buffer
A containing 0.5M NaCl at a flow rate of 200ml/h. Fractions
containing methyltransferase activity were combined (500ml) and
dialysed against 201 buffer B (lmM potassium phosphate pH 7.5,
lmM EDTA, 0.1% /3-mercaptoethanol, 10% glycerol) for 18h.
The dialysed sample was clarified by centrifugation at 15000xg
for 30 min and loaded on a single-stranded DNA cellulose column
(12cm x4cm diam.) equilibrated with buffer B. The column was
washed with 3 column volumes of buffer B and then eluted
successively with with 2 column volumes each of buffer B
containing 0.1M NaCl, 0.3M NaCl and 1M NaCl at a flow rate
of 40ml/h. The activity was eluted with 1M NaCl, although the
enzyme has typically been eluted with 0.3M NaCl with other
batches of DNA cellulose. The pooled active fractions (25ml)
were loaded onto a column of Ultrogel AcA54 (120 cmx2.5cm
diam.) equilibrated with buffer C (0.1M NaCl, 15mM potassium
phosphate pH 7.4, lmM EDTA, 0.1 % j3-mercaptoethanol, 10%
glycerol). At this NaCl concentration, the active fractions were
eluted at 1.3-1.5 times the void volume of the column (Vo),
which is earlier than previously reported (1.8x Vo) when buffer
C containing 0.5M NaCl was used. This altered elution behaviour
indicates possible aggregation of the methyltransferase or
interaction with other proteins at the lower salt concentration.
The active fractions were pooled and concentrated using an
Amicon ultrafiltration cell equipped with a Diaflo YM10
membrane. Total yield was about 800 pmole active enzyme
(0.5% recovery) with a specific activity of 300 units/mg protein
(500-fold purification). Incubation of the enzyme with
[3H]-labelled substrate followed by SDS-Page electrophoresis
and fluorography showed a major radioactive product of about
24kD (and a minor labelled species at about 27kD, probably
resulting from incomplete denaturation of the labelled protein)
in good agreement with previous estimates of the molecular mass
of the protein (17).
Labelling the Enzyme and Binding to a Solid Support
An estimated total of 600pmole partly purified enzyme in 6ml
buffer C was dialysed into reaction buffer by ultrafiltration using
a Diaflo YM10 filter and the volume adjusted to 20ml. A 2ml
aliquot of this preparation was incubated for 30min at 37 °C with
106 cpm of [3H]-labelled DNA substrate in a glass test-tube,
18ml was similarly incubated with lnmole m6-Gua-containing
oligonucleotide. The two reaction mixtures were then combined.
2.4g siliconized glass wool, prepared by immersion in Repelcote
(BDH) followed by several washes in distilled H2O, was then
added and the methylated enzyme was allowed to bind to the
glass wool by incubation for a further 30 min at 37 °C. The glass
wool was then washed 3 times in assay buffer and twice in
distilled H2O and allowed to drain without drying.
Trypsination and Size Fractionation
The washed siliconized glass wool containing the adsorbed
radiolabelled methyltransferase was immersed in 20ml lOmM
NH4HCO3 pH 8.1, lmM CaCl2, 0.05% Tween 20 containing
0.1/ig/ml trypsin and incubated at 20°C for 16 hours. The
trypzinised sample was concentrated by evaporation under
vacuum to a final volume of 1.1 ml and precipitated material was
removed by centrifugation. In order to separate intact trypsin from
the shorter peptides, the sample was applied to a column
(38cm x lcm diam) of Sephadex G25 (superfine) equilibrated with
lOmM NH4HCO3 pH 8.1. A symmetrical radioactive peak
which eluted at 1.3x the void volume was collected and
evaporated to dryness in a vacuum desiccator. This peak
contained approximately lOOpmole methylated peptide as
estimated from its [3H] content. This step also effectively
removed very short peptides.
Ion Exchange Chromatography (FPLC) of Peptides
The Sephadex G25-purified dried sample was dissolved in 0.5ml
buffer D (20mM 2[N-morpholino]ethanesulfonic acidNaOH pH
6.0, 0.02% Tween 20) and subjected to FPLC using a MonoS
cation exchanger (5cm x0.5cm diam). The flow rate was
0.5ml/min with a gradient from buffer D to E (buffer D
containing 0.4M NaCl) over 60 min. The main radioactive peak
Nucleic Acids Research 19
appeared at a NaCl concentration of 0.12M with a minor peak
at 0.14M. The fractions corresponding to the main peak of
methylated peptide (about 35 pmole) were further purified by
HPLC.
HPLC
The sample was prepared for HPLC by adsorption to a SEPPAK C-18 cartridge (Waters) equilibrated with 50mM NH4AC
pH 7.0, washed with 20% acetonitrile and the peptide eluted with
40% acetonitrile in the same buffer. The sample was then
lyophilised, dissolved in lO/il 70% formic acid, adjusted to 100/d
in Buffer F (1 % acetonitrile, 0.08% trifluoroacetic acid (TFA))
and applied to an Aquapore ODS C-18 column (22cm X0.2 lcm
diam., Brownlee Labs) equilibrated in buffer F. The column was
eluted at a flow rate of 0.4ml/min with a gradient of 0 - 6 0 %
buffer G (90% acetonitrile in 0.06% TFA) over 70min. lml
fractions were collected and the two fractions containing
radioactive material were retained and separately lyophilised.
Amino Acid Sequencing
The samples were dissolved in 30/tl 0.1 % TFA, adsorbed to
Polyprene-treated glass discs and sequenced using a ABI 477A
peptide sequencer. A portion of the sample from each Edman
degradation cycle was saved for radioactivity determination.
RESULTS
Purification of an Oligopeptide Containing the
Methyltransferase Active Site
In preliminary experiments (data not shown) to purify the
methylated form of the methyltransferase, we obtained extremely
poor yields of the protein using a variety of experimental
approaches. Although the methylated form of die protein does
not bind to glass, it exhibits an exaggerated tendency to adhere
to hydrophobic surfaces even in the presence of surface active
non-ionic detergents. Since this tendency to interact with
hydrophobic surfaces— in particular with siliconized glass— was
not shared by the contaminating proteins in the partially purified
material, we used this property to effect a further purification.
A quantitative recovery of the intact methylated protein after its
adsorbtion to a siliconized glass support could be achieved only
by the use of high concentrations of ionic detergent (1 % SDS),
the presence of which severely limited the possibilities for further
purification. In contrast, radioactivity from the specifically
labelled methylated protein could be recovered in >90% yield
following a digestion with trypsin in the presence of the nonionic detergent Tween 20. This observation formed the basis of
the purification of a tryptic peptide containing the active cysteine
residue of the methyltransferase.
Approximately 60pmole of partially (500-fold) purified
methyltransferase was incubated with [3H]-substrate DNA
containing 70 pmole [3H]m6-Gua and 540pmole enzyme was
incubated with an excess of non-radioactive m6-Gua-containing
double-stranded oligonucleotide. The reaction mixtures were
combined and supplemented with siliconized glass wool to which
the methylated enzyme was allowed to adsorb. Non-adsorbed
proteins were removed, along with the substrate DNA and
oligonucleotide, by extensive washing with assay buffer followed
by water. In preliminary experiments, SDS-PAGE and protein
determination were used to monitor the composition of the starting
material and the material which bound to glass wool and could
be removed by 1% SDS. This analysis indicated that >90% of
the contaminating proteins were removed in this step.
The radioactive protein was removed from the glass wool by
trypsin digestion. The glass wool bearing the methylated protein
was submerged in a solution containing 0.05% Tween 20 and
0.1/tg/ml trypsin and the mixture incubated at 20°C overnight.
The resulting tryptic digest was applied to a column of Sephadex
G25 to separate the trypsin from the oligopeptides. Trypsin eluted
in the void volume (VJ of the column whereas the radioactive
material was included and eluted as a symmetrical peak at
approximately 1.3xV 0 . This material was retained and further
fractionated by FPLC using a MonoS cation exchange column.
Figure 1 shows the elution pattern from the MonoS column. The
majority of the radioactive material eluted as a sharp peak at a
NaCl concentration of 0.12M with a small shoulder appearing
at around 0.14M NaCl.
Fractions from die major radioactive peak eluting at 0.12
MNaCl were pooled and further purified by reverse phase HPLC
using an Aquapore ODS C-18 column. This procedure resolved
the complex mixture of peptides into a number of peaks which
absorbed at 220nm (Figure 2). However, all the radioactivity
was recovered in two fractions coincident with a single peak of
absorbance. The two radioactive fractions which contained
respectively 12 and 6pmoles of methylated peptide (representing
an overall yield of 3%) were separately subjected to amino acid
sequence analysis.
Amino Acid Sequence of the Active Site Peptide
The amino acid sequence of the purified peptide from the fraction
from the HPLC column containing 12pmole is shown below:
1 (?) • 2. Asn • 3. Pro 4. He 5. Pro • 6. He (Phe)
7. Leu (Asp) 8. Thr (He) 9. Pro (Gin)
The sequence obtained from the fraction containing 6 pmole of
peptide provided confirmation of this sequence. In the later
sequencing cycles, corresponding to weaker signals, more than
one amino acid was detected indicating the presence of a low
level of contaminating oligopeptide. Where two amino acids were
detected, both are shown. In each case, however, the first is the
more likely as judged by its relative abundance. Methylcysteine
yields very poor fluorescence following derivatisation and is not
detectable under the standard conditions used for automated
sequencing. A portion of the sample from each Edman
degradation cycle was therefore retained and its
[3H]-radioactivity determined separately. Figure 3 shows that no
1
20
40
60
Fraction No
Figure 1. Purification of the Radioactively Labelled Active Site Peptide by FPLC.
Separation was carried out on a MonoS cation exchange column. Elution buffers
contained: 20mM MES NaOH pH 6.0, 0.02% Tween 20 and a gradient of NaCl
(0-0.4M) as shown. Fractions (0.5ml) were collected and aliquots (lO/il) were
taken for radioactivity determination. Fractions 26—28 were pooled and used
for further purification.
20 Nucleic Acids Research
2
Ada
Ogt
Asn
Asn
Dat1
Asn
Bovine Asn
3
4
5
Leu Ala
Pro | lie | Ser
Asp Leu Pro
Pro | lie | Pro
6
7
8
9
10
11
12
13
lie
lie
lie
Val
Val
Phe
lie
Val
Val
Pro
Pro
Pro
His
His
His
Arg
Arg
Art,
Val
Val
Val
Me
Leu
Thr
Pro
Cys
Cys
Cys
Cys
Figure 4. Comparisons Among the Active Sites of O6-Methylguanine-DNA
Methyltransferases.
Ada: The C-terminal O6-Methylguanine-DNA methyltransferase active site of
the E. coli Ada protein.
Ogt and Datl: The putative active sites of the E. coli Ogt and the B. subtilis
Datl proteins.
Bovine: The sequence of the purified calf thymus peptide.
The methyl accepting cysteine is arbitrarily assigned position 10. Amino acids
exhibiting homology to the bovine sequence are shown boxed.
DISCUSSION
20
40
Fraction No
Figure 2. Reverse Phase HPLC Purification of the Labelled Peptide. Separation
was performed on an Aquapore ODS C-18 column (22cmxO.2 lcm diam.). The
column was eluted with a linear gradient (0-60%) of 90% acetonitrile/0.06%
TFA in 1% acetonitrile/0.08% TFA. The flow-rate was 0.4ml/min over 70min.
The absorbance baseline has been arbitrarily set at the bottom of the Figure.
Fractions (1 min) were collected and the radioactivity in aliquots was determined.
The total radioactivity present in fractions 31 and 32 is indicated by the bars.
All other fractions contained only background radioactivity. The two radioactive
fractions were independently subjected to amino acid sequence analysis.
•
ctivit
do) A
"E
100
(0
o
I
±
II
150
50
•
1
I
/I
/
I
\
\
10
15
20
Cycle No
Figure 3. Localization of the S-[3H]-methyl Cysteine Residue in the Peptide.
Fraction 31 from the ODS C-18 column was subjected to automatic amino acid
analysis. The radioactivity in aliquots of the fractions from each Edman degradation
cycle from the automatic peptide sequencer was determined by liquid scintillation
counting.
radioactivity was released before the 10th sequenator cycle. More
than 60% of the radioactivity was recovered in the 10th cycle.
The remaining <40% was eluted in the 1 lth cycle. These data
strongly indicate that the acceptor cysteine residue of the
methyltransferase follows the proline residue at position 9 in the
above sequence.
In Figure 4 we present a comparison of the sequence of the
m6-Gua methyltransferase active site of the Ada protein of
E. coli along with the proposed active sites for two other related
methyltransferases; the constitutive Ogt protein of E. coli and
the B. subtilis Datl protein. The data reveal a high degree of
conservation in the protein sequence around the active sites of
these proteins. The conserved amino acids in the bacterial
enzymes; the asparagine at position 2, isoleucine at position 6
and proline at position 9 (the methyl acceptor cysteine is
arbitrarily assigned position 10) are also conserved in the bovine
enzyme. It is of further interest that the amino acids on the Nterminal side of the acceptor cysteine are of a predominantly
hydrophobic nature. In this comparison, we have used the
experimentally determined most probable amino acids for
positions 5—9 of the bovine enzyme. In the case of the leucine
residue at position 7, this assignment is also supported by the
observation that the radioactive tryptic peptide bound to a MonoS
cation exchange column but not to a MonoQ anion exchange
column. This behaviour is not compatible with the presence of
a negatively charged amino acid, aspartic acid, at position 7.
However, if as seems likely, the His-Arg doublet adjacent to the
acceptor cysteine in the other three methyltransferases is
conserved in the bovine enzyme, this would introduce two
positively charged amino acids into the peptide. A peptide of this
sequence would still retain an overall positive charge with Asp
assigned to position 7 and would be expected to exhibit the
observed behaviour on ion exchange chromatography. We note,
however, that the presence of Tween 20 is necessary to prevent
irreversible binding of the peptide to both the anion and cation
exchangers, most probably as a result of hydrophobic interactions.
For this reason, we consider the assignment of Leu to position
7 to be most likely since it is more compatible with the
hydrophobic nature of this region of the peptide. While the
assignment of isoleucine to position 8 would represent a closer
homology to the sequence of the E. coli Ada protein, the overall
hydrophobic nature of the amino acid sequence preceding the
acceptor cysteine would nevertheless be maintained by the
presence of a threonine residue in this position.
The substantial homology between the active sites of these
proteins probably reflects a conservation of their reaction
mechanism. The Datl protein and the Ogt protein exhibit
considerable homology (approaching 50%) elsewhere in the
primary sequence. Similarly, the carboxy-terminal domain of the
Ada protein which contains the m6-Gua methyltransferase active
site is highly homologous to both the Ogt and Datl proteins
(13,14). In fact, a C-terminal fragment of the Ada protein which
Nucleic Acids Research 21
is similar in size to both Ogt and Datl can function efficiently
as an m6-Gua methyltransferase in its own right (20).
Despite the close resemblances among the bacterial protein
sequences and the similarity we have observed at the active site
region of the bovine enzyme, it seems likely that such a high
degree of conservation between the prokaryotic and mammalian
enzymes around the active site of the methyltransferase merely
reflects the nature of the methyltransfer reaction, in particular
the mobilisation of the methyl group, and is unlikely to extend
throughout the protein. In a recent comparison of the known
sequences of methyltransferases which catalyse the methylation
of cytosine residues in DNA, Posfai et al (21) observed that
a ProCys doublet was present in a block of relatively invariant
amino acids around the putative active site of the cytosine
methyltransferases. It is of interest that, in the cytosine
methyltransferases, the amino acid preceding the ProCys
doublet by five amino acids is either Leu or He. Furthermore,
the ProCys doublet is preceded by a block of hydrophobic
amino acids. Both of these structural features are present in the
active site region of the m6-Gua-DNA methyltransferases.
Thus it seems likely that the particular energetic requirements
of methyl group transfer will impose a strong selective pressure
for conservation of the active site region of the
methyltransferases. Such selective pressure may not apply to
the remainder of the protein sequence. In support of this idea
is the observation that both monoclonal antibodies and polyclonal
antisera raised against the purified C-terminal domain of the
E. coli Ada protein, which bears the m6-Gua DNA
methyltransferase function, do not cross-react with the human
or bovine methyltransferases on immunoblots (J.Hall,
B. Sedgwick, unpublished data).
An unambiguous assignment of the active cysteine residues
of a suicidal methyltransferase has so far only been made for
the two domains of the E. coli Ada protein (22,23). While the
conserved sequences among the other bacterial enzymes strongly
suggest the location of their active sites, direct evidence is
lacking. The establishment of the active site sequence of the
bovine methyltransferase reported here will, despite its
ambiguities, enable an unequivocal assignment of the active site
to be made when the full sequence of the protein becomes
available.
ACKNOWLEDGEMENTS
We thank Claire Stephenson for skilled technical assistance, Iain
Goldsmith for oligonucleotide synthesisis and Ron Brown for the
amino acid sequence analysis.
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