Download Purification to homogeneity and partial amino acid sequence of a

© 1990 Oxford University Press
Nucleic Acids Research, Vol 18, No 6
1351
Purification to homogeneity and partial amino acid
sequence of a fragment which includes the methyl
acceptor site of the human DNA repair protein for
C^-methylguanine
G.N.Major*, E.J.Gardner1, A.F.Carne2 and P.D.Lawley
Alkylation Carcinogenesis Team, Chemical Carcinogenesis Section, Institute of Cancer Research,
Chester Beatty Laboratories, Fulham Road, London SW3 6JB, UK, 1CRC Unit of Human Cancer
Genetics, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2
1QP and department of Protein Chemistry, Celltech Ltd., Bath Road, Slough SL1 4EN, UK
Received January 19, 1990, Revised and Accepted February 27, 1990
ABSTRACT
DNA repair by O6-methylguanine-DNA methyltransferase (C^-MT) is accomplished by removal by
the enzyme of the methyl group from premutagenic
O6-methylguanine-DNA, thereby restoring native
guanine in DNA. The methyl group is transferred to an
acceptor site cysteine thiol group in the enzyme, which
causes the irreversible inactivation of O^-MT.
We detected a variety of different forms of the
methylated, inactivated enzyme in crude extracts of
human spleen of molecular weights higher and lower
than the usually observed 21 -24kDa for the human
O6-MT. Several apparent fragments of the methylated
form of the protein were purified to homogeneity
following reaction of partially- purified extract enzyme
with O6-[3H-CH3]methylguanine-DNA substrate. One of
these fragments yielded amino acid sequence
information spanning fifteen residues, which was
identified as probably belonging to human
methyltransferase by virtue of both its significant
sequence homology to three procaryote forms of
C^-MT encoded by the ada, ogt (both from E. coll) and
dat (B. subtilis) genes, and sequence position of the
radiolabelled methyl group which matched the position
of the conserved procaryote methyl acceptor site
cysteine residue. Statistical prediction of secondary
structure indicated good homologies between the
human fragment and corresponding regions of the
constitutive form of O*-MT in procaryotes (ogt and dat
gene products), but not with the inducible ada protein,
indicating the possibility that we had obtained partial
amino acid sequence for a non-inducible form of the
human enzyme. The identity of the fragment sequence
as belonging to human methyltransferase was more
recently confirmed by comparison with cDNA-derived
amino acid sequence from the cloned human O*-MT
gene from HeLa cells (1). The two sequences compared
* To whom correspondence should be addressed
well, with only three out of fifteen amino acids being
different (and two of them by only one nucleotide in
each codon).
INTRODUCTION
C^-methylguanine (C^-MeG) is a potent premutagenic lesion
that may be formed in DNA following reaction with a methylating
carcinogen (2). In animal models, the presence in this base lesion
in DNA is positively correlated with both tumour and
autoimmune disease induction (3), and a point mutation of the
type expected from C^-MeG was found to cause the malignant
activation of the cellular H-ras oncogene in methylnitrosourea
(MNU)-induced rat mammary tumours (4). A repair activity for
C^-MeG was first described in E. coh by Lawley and Orr (5),
and subsequently in other procaryotes and eucaryotes, including
mammals (for reviews, see refs 6—8). This repair is performed
by the enzyme C^-MT, that acts by removing the methyl group
from the O6 atom of guanine, thereby restoring native guanine
in DNA, and transferring it to an internal cysteine residue (9,10).
The methylated enzyme is inactivated as a consequence of this
reaction and is not regenerated (9,10). In E. coli, (AMT also
repairs C^-methylthymine and the S-stereoisomer of
methylphosphotnesters in DNA (11), substrate specificities that
have largely not been detected in association with mammalian
C^-MT (12,13).
Multiple forms of C^-MT have been found in E. coli (14,15)
and B subtilis (16,17) that differ both in substrate specificities
other than C^-MeG, and inducibility following exposure of cells
to low levels of a methylating carcinogen such as jV-methylyV'-nitro-N-nitrosoguanidine (15-17). The induced form of
0^-MT in E. coli (the ada gene product) is able to be cleaved
by cellular protease action to smaller catalytically active
polypeptides, each displaying particular substrate specificities
(18,19). Genes encoding the multiple forms of C^-MT in E. coli
and the constitutive form in B. subtilis have been cloned and
1352 Nucleic Acids Research
sequenced (14,16,20), and with the exception of the dot gene
product, the proteins expressed by these genes purified to
homogeneity and characterised (21,22). By comparison,
mammalian C^-MT has remained poorly characterised Over the
past 8 years, a number of investigators have reported protocols
for the partial purification of Cfi-MT from both human (23-26)
and rat (10,27) tissues. Many have encountered technical
difficulties during the purification of the enzyme. A major
hindrance to the isolation of C^-MT in pure form has been its
low abundance in mammalian tissues. A more refractory difficulty
in the study of the enzyme has been its high degree of instability
upon purification.
We report a procedure for the purification to homogeneity of
fragments of the methylated form of C^-MT. This procedure
serves to reduce or avoid the problems enumerated above and
permitted us to obtain amino acid sequence information for the
methyl acceptor site region of the human enzyme.
MATERIALS & METHODS
Reagents
[ 3 H-CH 3 ]Methylnitrosourea ([ 3 H]MNU) at a specific
radioactivity of 1 Ci/mmol and [14C]-radiolabelled protein
molecular weight markers were from Amersham International.
High molecular weight calf thy mus DNA, bovine serum albumin
(protease-free) and protease K (type XI; DNase-free) were from
Sigma, and with the exception of cetyltrimethylammonium
bromide (CTAB) which was reagent grade, EDTA, calcium
chloride and all inorganic solvents were purchased as analytical
reagent grade materials and all organic solvents were HiPerSolv
HPLC grade, from BDH, Dorset, U.K Highly pure dithiothreitol
(DTT) was from Calbiochem, and Ultrapure grade Tris and
ammonium sulphate were from Gibco-BRL All electrophoresis
reagents and prestained and unmodified protein markers were
from BioRad. All other reagents (of analytical grade) and
enzymes were from Sigma.
Human Spleen Extracts
Human spleens from patients undergoing splenectomy for
idiopathic thrombocytopoenia purpura, Hodgkin's or nonHodgkin's lymphoma, were obtained by courtesy of Dr. R.L.
Carter, Section of Pathology, Royal Marsden Hospital, Sutton,
U.K., and usually within 30 nun of organ removal. The tissue
was sectioned, frozen on dry-ice for transportation to the
laboratory, then stored at -75°C if not used straight away.
Whole tissue extracts were routinely prepared from 70g of spleen,
essentially by the method of Pegg et al (10)
C'-MT activity was concentrated and partially-purified from
this extract by precipitation with a final amount of 50% (at 4°C)
ammonium sulphate. Protein precipitates, which contained all of
the recoverable C^-MT activity, were collected by centrifugation
at 143,000xg (max.) for 40 min at 4°C, redissolved in 240 ml
of 50 mM Tris/HCl, pH 8.3 (at 22°C), 0.5 mM EDTA, 1 mM
DTT (buffer A), and dialysed for 18h at 4°C against three 20
vol changes of the same buffer. The dialysed extract was clarified
by centrifugation as above for 60 min and collected as a 250 ml
supernatant.
Q-Sepharose Fast-Flow Anion-exchange FPLC
Using an FPLC system, a water-jacketed XK 50/30 column
(Pharmacia-LKB) was packed with 300ml of Q-Sepharose FastFlow anion-exchange chromatography medium, and equilibrated
with 10 vols of buffer A, with the column cooled to 5°C. Partially
purified extract from the previous step was applied to the column
which was then washed with 400ml of buffer A to remove
unbound, non-specific protein. Enzyme activity was eluted from
the column by application of the following: (a) 1200 ml of buffer
A at the lower pH (at 22°C) of 7.13 (eluate collected in 8 X150
ml fractions), followed by (b) 850 ml of buffer A, at a higher
than previous pH of 7.85 (buffer B), made 50mM with NaCl,
(collected as a 250 ml, followed by 4 x 150 ml fractions), then
(c) stepwise elution with buffer B containing higher concentrations
of NaCl (see Fig.2).
Radiolabelled methylated DNA substrate
DNA containing O^-MeG with a radiolabel in the methyl group,
was prepared for use as an C^-MT assay substrate by the
method of Demple et al. (28). Tritiated, methylated DNA
substrate ([3H]MeDNA) prepared in this manner was
additionally dialysed twice against 200 vols of buffer A, then
subjected to methylpurine analysis by cation-exchange HPLC
(following hydrolysis of the DNA in 0.1M HC1) using the method
of Lawley et al. (29), and routinely found to contain 55-75%
of its radiolabelled methyl groups in association with C-MeG.
Assay of 0*-MT
For the assay of C^-MT, we used a modification of a previously
suggested method for measuring the in vitro repair of
C^-ethylguanine in DNA (30). Our assay, a detailed description
of which will be reported elsewhere, is an adaptation of
Schneider's method (31) for the measurement of DNA following
its precipitation by CTAB in the presence of proteinase K, the
latter removes any protein remaining in association with DNA.
The assay incubation mixture contains enzyme extract made to
200 n\ with buffer A, which is added to 10 fi\ of [3H]MeDNA
(routinely used at a level of 4-5000 dpm/10 /*1). The repair
reaction was performed to completion by incubating the assay
mixture in a water bath for 90min at 37°C, followed by the
addition and mixing on ice, of 300 /tl of 80 mM EDTA, pH 6.0,
containing 200 ng of calf thymus DNA. All DNA is then
separated from methylated, radiolabelled protein by precipitation
of the former with the addition and mixing of 200 /tl of 3%
CTAB. Labelled protein left in association with DNA is
solubilized by subsequent addition of and incubation for 60 min
at 37°C with 10 n\ of freshly prepared lmM CaCl2 containing
50 ng of proteinase K. This mixture is then spun at 15,000xg
for 15 min in a micro-centrifuge to pellet the DNA Radioactive
methyl groups associated with protease-digested protein were
measured in a 650 /il sample of the resultant supernatant by
scintillation counting.
The stoichometric reaction between O'-MT and
C^-methylguanine-DNA substrate permits the calculation of
enzyme molar amount from knowledge of the specific
radioactivity of the [3H]MNU used to make the methylated
DNA substrate. Very approximate molecular weights were
derived for homogeneous C^-MT fragments from knowledge of
their molar amount (assuming one radiolabelled methyl
group/enzyme fragment molecule) and crude estimation of their
gravimetric amount from their integrated absorbance at 214nm
(assuming an A2]4 of 15, 1 = lcm, for a protein solution of
concentration lmg/ml).
Methylation and inactivation of C^-MT for subsequent
purification
Peak I enzyme activity fractions from Q-Sepharose Fast Flow
(see above) were pooled as indicated in Fig.2. The pooled
Nucleic Acids Research 1353
Table 1. Purification of 06i-methylguarune-DNA methyltransferase from 70 g of human spleen
Fraction
Crude extract
ammonium sulphate
Q-Sepharose Fast Flow1
Hydroxyapatite
Total activity
(pmol)
Total protein
(mg)
Specific activity
(pmol/mg)
Purification
(n-fold)
Yield
(*)
4301
(4464)
2686
(3291)
1118
(1207)
9512
(362*)
4116
(4886)
1190
(1714)
10
(31)
40
(0 25)
10
(0.9)
23
(19)
111
(39)
238
(1448)
10
(10)
22
(2 1)
107
(43)
229
(1591)
100
(100)
62
(74)
26
(27)
22
(8)
Figures in brackets indicate a separate purification experiment which utilised hydroxyapatite column chromatography method 1, while those
figures not in brackets were from an experiment using hydroxyapatite method 2 (see accompanying text and 'Methods' for details).
'Peak I fraction
2
Figure based on amount of [3H-CH3]methylated and inactivated C^-MT present in fraction
fractions were reassayed for enzyme activity to determine the
minimum amount of [3H]MeDNA that would be required to
methylate all of the peak I C^-MT, which was subsequently
performed using an incubation, with occasional stirring, at 37°C
for 60 min.
Hydroxyapatite column separation of methylated Cfi-MT
and DNA
Inactivated, [3H-CH3]-methylated protein from the previous step
was separated from repaired DNA by hydroxyapatite
chromatography by either of two methods, as follows:
Method 1: the methylated protein-DNA mixture was dialysed
against 3x20 volume changes of lOmM sodium phosphate
buffer, pH 6.8 (buffer C), over 18h at4°C. The dialysed sample
was applied to the top of a 50 ml bed volume column (in a
Pharmacia XX 26 column) of hydoxyapatite (pre-equilibrated
with 10 volumes of buffer C), at a flowrate of 0.5ml/min. Upon
completion of sample application, the column was washed with
buffer C until the A ^ of the eluate returned to baseline. [3HCH3]Methylated C^-MT was both separated from residual
substrate [3H]MeDNA and eluted from hydroxyapatite, by
application of a 0 - 2 M gradient of guanidium hydrochloride in
550ml of buffer C, made lOmM with DTT (Fig.4a). DNA
remaining on the column could be subsequently eluted by
application of a gradient of 10-480mM sodium phosphate buffer,
pH 6.8 (data not shown).
Method 2: the methylated protein-DNA mixture was directly
adsorbed to 20g of solid hydroxyapatite for 30min at ambient
temperature. The resultant slurry was then used to make a column
of similar dimensions to that used in Method 1. This column was
washed and eluted as for Method 1, with the exception that an
FPLC system was used to pump washing buffer and the
guanidinium hydrochloride gradient onto the column, at a
flowrate of 2 ml/min.
Reversed-phase chromatography of methylated protein
[3H-CH3]Methylated protein, that eluted from the above
hydroxyapatite column, was pooled and further purified by the
sequential use of reversed-phase (RP) FPLC and RP-HPLC. A
'protein' RP column (Ci/C8; HR 10/10 semi-preparative scale
column; Pharmacia-LKB) was used for RP-FPLC and run with
a flowrate of 2 ml/min, at a room temperature maintained
between 15 and 20°C. A Ci& non-porous resin (NPR) column
(Octadecyl-NPR; 4.6 mm I.D.X3.5 cm, Tosoh Corp., Japan)
was used for RP-HPLC, which was performed using a Water's
HPLC system, at a flowrate of 1.5 ml/min and at the elevated
room temperature of 30°C which, for repeated RP-HPLC of
homogeneous methylated protein, increased recoveries from
5-10% at the lower ambient temperature to 85-100% at 30°C.
RP chromatography was performed using either 0.1%
trifluoroacetic acid (TFA; Pierce) or 0.1 % heptafluorobutyric
acid (HFBA; Pierce) as pump A solvent, in combination with
linear gradient elution using 70% acetorutrile in solvent A (solvent
B). Eluate absorbance was monitored at 214 run.
A summary of the RP chromatography purification procedure
and conditions used is depicted in Table 2.
Amino add sequence determination
Automatic sequencing of apparently homogeneous C^-MT
polypeptides was performed with a gas-phase sequencer (Applied
Biosystems, model 470A) with on-line detection of the PTHamino acids by HPLC using a gradient elution system (Applied
Biosystems PTH-analyzer, model 120A). Polypeptides were
dissolved in 20-80 /tl of 0.1 % TFA and spread, in 20 /J aliquots,
into a glass fibre disk which was precycled three times with 20
/xl of an aqueous solution containing 0.5 mg of BioBrene Plus
(Applied Biosystems) and 33 /ig of sodium chloride.
Detection of tritiated protein in gel slices and electroblots
following SDS-PAGE
Indicated fractions containing [3H]methylated C^-MT were
subjected to discontinuous SDS-PAGE in 15% acrylamide gels,
by the method of Laemmli (32). Test fractions were duplicated
in each gel and following electrophoresis, gel lanes run with one
set of test were sectioned into 3mm slices, then radiolabelled
protein was extracted from these slices and measured by
scintillation counting essentially as described previously (33),
while duplicate lanes were electroblotted onto nitrocellulose
(0.1 /tm, from Schleicher and Schuell) following the method of
Towbin (34) using a 20h electrotransfer at 4°C but half the
suggested strengui of transfer buffer to reduce heating in die
electrotransfer system. Radiolabelled protein bands on electroblots
were visualised by fluorography using the method of Roberts (35)
using Fuji RX film.
Protein assay
Protein was determined by die method of Bradford (36) using
a BioRad protein assay kit and BSA as a calibrating protein
standard.
1354 Nucleic Acids Research
TaMe 2. Reversed-phase chromatographyli punfication scheme for [3H-CH3 Jmethylated C^-MT peptides
Chromatography procedure
iColumn
Packing alkyl
chain length
Step
Type
1
ProRPC C,/C,
HR 10/K)
Solvent A
Solvent B
for sample
loading
0 1 % TFA
0%
0-80%
in 160 nun
resolves [3H-CH3]methylated
peptides from hydroxyapatite
step (see 'Methods' section)
0.1% HFBA
15%
15-25% B
in 2 mm
performed with 1 peak of
radiolabelled peptide from
step 1
Solvent B gradient
for elution
(FPLO
2
NPR
(HPLC)
C,8
Comment
Example peptides3
25-50% B
in 20 rrun
3a2
NPR
(HPLC)
C,g
0 1 % TFA
15%
15-35% B
in 16 min
used with peptide (from
step 2) of lesser
hydrophobicity
CRC1/2 and
CRC2/2
3b2
NPR
(HPLC)
c,»
0 1% TFA
20%
20-30% B
used with peptide (from
step 2) of greater
hydrophobicity
CRC3/2 and
CRC4/2
30-40% B
in 10 nun
' see 'Methods' section for further details
Peak fractions of radioactive peptide punfied through this step were at least twice, rechromatographed, using these conditions
see also Fig 5
2
3
RESULTS
A summary of the purification procedure, including the use of
two moderately different hydroxyapatite chromatography
methods, is given in Tables 1 and 2.
The most interesting but unexpected finding along the route
to purification to homogeneity of C'-MT was the detection of
different forms of the [3H-CH3]methylated, inactivated enzyme
in partially purified human spleen extract, which included forms
larger than the single ~24kDa enzyme usually observed in
similarly prepared and methylated extracts of mammalian tissues
(see 'Introduction') (Fig. 1). The major electroblotted band
detected by fluorography displayed an apparent molecular weight
of 34kDa, and the minor bands, which showed a parallel decrease
in apparent amount and size, migrated in SDS-PAGE with
molecular weights of 30kDa, 24kDa, 20kDa, 19kDa, 17kDa and
15kDa, which suggested that degradation of the methylated,
inactive protein had occurred in the human spleen extract. As
expected, presumed enzyme degradation, by endogenous
proteases, and the generation of an array of molecular weight
forms of methylated C^-MT lower than 34 kDa (Fig. 1) were
routinely found to be more extensive in extracts made for
purification work than in similar extracts that were prepared on
a much smaller scale (data not shown).
The question of whether or not inacti vation upon methylation
of C^-MT primes the enzyme for degradation by proteases was,
in part, answered by the finding of two apparently different forms
of the active enzyme that were resolved in roughly equal amount
by anion-exchange chromatography (Peaks I and D, Fig.2). Peak
I enzyme was also detected, but in much less amount relative
to the peak n enzyme, in the above small-scale extracts of human
spleen, and was completely resolved from peak II enzyme using
salt gradient elution of enzyme activity from a smaller (Mono
Q) anion-exchange column (results not shown). This change in
relative yields of the different forms of C^-MT activity between
small-and large-scale extracts suggests that peak I enzyme is
produced from peak II enzyme as a result of protease digestion
of the latter. To further establish that the purification-scale peaks
I and II forms of C^-MT were different and not due to a
chromatographic artifact, we examined the thermal stability of
each enzyme to preincubation at 45 °C prior to enzyme activity
assay. The results in Fig.3 show that the peak I enzyme is more
temperature-labile than the peak II protein, which indicated that
the differently eluting activities represent different forms of
C'-MT. The broadness of eluted peak I activity (Fig 2) in
combination with its temperature lability relative to peak II
enzyme, suggests that peak I material may comprise a
heterogeneous collection of catalytically active C^-MT
fragments of different size. Overall, therefore, it appears that
protease degradation of C^-MT, which was more evident
following larger-scale enzyme extraction from tissue, occurred
early in the in purification procedure, and produces catalyticaJly
active fragment forms of a larger enzyme.
Given these findings and other workers' experienced difficulties
with regard to the purification of active mammalian C'-MT, we
chose to alkylate, and hence inactivate, enzyme purified through
the anion-exchange the chromatography step, and perform
subsequent purification with the [3H-CH3]-methylated form of
C^-MT whose detection through subsequent purification steps
did not rely upon preservation of enzyme activity. Peak I enzyme
was selected for this work due to its much greater degree of purity
than Peak II activity. Enzyme eluting under peak II (Fig. 2) was
not further characterised.
Following reaction with [3H]MeDNA substrate, [3HCH3]methylated, inactivated peak I enzyme was separated from
residual DNA by hydroxyapatite chromatography (Fig. 4).
Batchwise adsorption of the reaction products to hydroxyapatite
prior to column construction and elution, affords, in addition to
separation from DNA, a modest purification of C^-MT and its
recovery in good yield (Fig.4b; Table 1). Conversely, direct
sample application to a column of hydroxyapatite generated an
enzyme preparation of much higher purity, but of poor yield (Fig.
Nucleic Acids Research
1355
(a)
(b)
JL
200-,
-1000
1 1 1 1 1
600r
in.
150 0 8
oe
-100
100 Q.
•o
O
f
04
20G
50•O-2
d.f.
I
14
27
J-o
n
10
40
Elutlon Time (mm x 10 )
Gel Slice Number
Fig. 1. SDS-PAGE and detection by fluorography of different forms of [3HCH3]methylated O'-MT [l4C]-radiolabelled calibrating proteins are myosin
(200kDa), phosphorylase b (92 5 kDa), bovine serum albumin (69 kDa), ovalburrun
(46 kDa), carbonic anydrase (30 kDa), trypsin inhibitor (21 5 kDa) and lysozyme
(14 3 kDa) o , origin, d f , dye front The detection of the different enzyme
bands by fluorography, (a), required a 90-day exposure against X-ray film This
long exposure time produced fluorograms of barely suitable quality for
photographic reproduction and, as such, faint enzyme bands originally seen are
indicated schematically The lower resolution, but much more rapid, method of
gel slicing (b) did not clearly define the presence of different enzyme forms
Recoveries of labelled protein in gel slices routinely varied between 26-35%
4a; Table 1). Given the very low abundance of C^-MT relative
to other proteins (and despite human spleen being a comparatively
rich source of enzyme), which was magnified in the present study
by the detected presence of an array of fragment forms of the
enzyme that eluted from hydroxyapatite (see below), we routinely
used the higher yield, hydroxyapatite method 2 (see 'Methods'
section). In the present study most of our preliminary enzyme
isolation work was, however, based on the use of the lower yield
but higher achieved enzyme purity method 1, which is only
recommended for use in this purification procedure if performed
on a — 4 times greater scale with respect to amount of starting
tissue used
Reversed-phase chromatography of methylated enzyme purified
through the hydroxyapatite step enzyme revealed the presence
of many [3H-CH3]methyl group-containing polypeptides. This
further suggested that the peak I fraction of active enzyme eluting
from the anion-exchange column contained an array of enzyme
fragments of differing size. Purification to homogeneity of
peptides containing the methyl acceptor site proved difficult. A
successful procedure was developed, however, based on the
Fig. 2. Q-Sepharose Fast Flow anion-exchange FPLC of partially purified
Cr-MT in human spleen extract Sample application and different column elution
(stepwise) conditions are indicated at the top of the figure (I) and (IT) are enzyme
activity peaks I and II (see 'Methods' section and accompanying text for further
details)
sequential use of reversed-phase media of differing
hydrophobicity (C8 and C18), two ion-painng reagents (TFA and
HFBA), and end-stage repeated use of a high resolution, nonporous C,8 reversed-phase HPLC column. A detailed summary
of this procedure is shown in Table 2, and by routine use of this
strategy we purified a series of C^-MT fragments of apparently
different size and hydrophobicity to either near homogeneity or
to apparent homogeneity. The result of one such peptide isolation
and purification procedure is depicted in Fig. 5, where four
fragments ranging in molecular weight from 4—20kDa were
isolated in pure form. Several such polypeptides were subjected
to gas-phase protein sequencing, but only one, peptide CRC2/2,
provided useful amino acid sequence information (Fig. 6a).
During the successful sequencing of peptide CRC2/2, the amino
acid signal became barely discernible by cycle 14. However,
scintillation counting of an automatically collected sample (one
sixth of the total) of the PTH-amino acid produced at each cycle
of Edman degradation revealed the presence of the methyl
acceptor cysteine at position 15, which was detected in predicted
yield and with an expected radioactivity profile (Fig.6b). The
sequence of CRC2/2 was identified, at the time, as probably
belonging to C^-MT by virtue of its only significant homology
being to corresponding (Amethylguanine methyl acceptor site
sequences from three procaryote forms of the enzyme, the ada
(20,37) and ogt (14) gene products in E. coli and the dot gene
product (16) from B. subtilis (Fig.7). Confirmation of the identity
1356 Nucleic Acids Research
100
o
X
2 O
15
30
45
60
Preincubation Time (min)
Fig. 3. Effect of preincubation at 45°C on the activity of two forms of O'-MT
resolved by anion-exchangc chromatography (peaks I and II). Prior to pooling
fractions eluting as peaks I and n, as indicated in Fig 2, samples of the most
active fractions in each peak were preincubated for indicated times at 45 °C then
assayed for C^-MT activity. As it contained less protein than peak II, the peak
I sample's protein concentration was adjusted to that of the former by the addition
of bovine serum albumin (BSA, protease-free) Peak I activity (A), Peak II activity
(•)
of this sequence was provided by comparison with cDNA-derived
amino acid sequence from the recently cloned human C^-MT
gene from HeLa cells (1; Fig.7a), where only 3 differences in
amino acid assignment between the two sequences were found.
Fig.7b shows the great similarity between the secondary
structures, statistically predicted by the Chou and Fasman
technique (38), of the CRC2/2, HeLa and ogt sequences, but
to a progressively lesser extent with the dot and ada sequences.
DISCUSSION
An unexpected finding in the course of our purification work
was the detection in human spleen extracts an array of different
molecular forms of alkylated C^-MT with molecular weights
both higher and lower than the usually reported range of
21 -24kDa for a single form of this enzyme similarly extracted
from a variety of different human sources (23,33,39-41). The
most abundant form detected in partially purified extracts had
an apparent molecular weight of 34kDa, while the remaining
forms, which were of lower molecular weight and present in
lesser amount, displayed a parallel decrease in abundance and
size. This pattern might simply be due to the 34kDa enzyme being
partially degraded into lower molecular weight forms as a result
of endogenous protease action, and in support of this was our
finding of fewer different forms of C^-MT from tissue extracts
12
18
Fraction Number
Fig. 4. Separauon of [3H-CH3]methylated, inactivated C^-MT from residual
[ 3 H]MeDNA, following repair reaction, by hydroxyapatite column
chromatography (a) method 1, and (b) method 2 (see 'Methods' section for
details) Fractions eluting between arrows were pooled for further purification
prepared on a much smaller, analytical scale, in which protease
action is not expected to be as extensive.
That methylation of C^-MT is the signal for the enzymic
cleavage by protease action was to some extent ruled out by our
finding of two forms of the active, unmethylated enzyme with
different temperature stability (anion-exchange chromatography
peaks I and II), and the suggestion that activity eluting as peak
I was comprised of a series of catalytically active fragments
formed by digestion of a larger form of the enzyme (peak II)
by proteases. Fragmentation of the enzyme by proteases may have
been enhanced by inclusion in our enzyme buffers of DTT to
stabilize C^-MT activity (27) and to protect the reactive thiol
group of the enzyme's methyl acceptor cysteine, as suggestive
evidence was recently found for mediation of cleavage of the
inducible form of C^-MT , in E. coh, (the ada gene product)
to smaller polypeptides by a cellular cysteine protease (19), a
class of proteases some of which are known to be activated by
DTT in mammalian cells. An interesting possibility, for which
there exists no direct evidence, is a role for ubiquitin in the
proteolytic digestion and generation of different forms of
C'-MT. Conjugation to ubiquitin is generally regarded as a
method of targeting proteins for protease digestion (42). The rad6
DNA excision repair gene in yeast encodes a ubiquitinconjugating enzyme, and this association strongly implies that
ubiquitin is involved elsewhere in repair (43).
In addition to fragments of a larger enzyme, some of the
different methylated &-MT forms resolved by SDS-PAGE in
the present study may represent distinct molecular forms of the
Nucleic Acids Research
1357
(a)
(a)
5 6 pmol
- 4kDa
50
-val-(Aap)-Ala-Met-Arg-Gly-A8n-Pro-Val-X-lle-(Ala)-(lle)-
20
((Pro)HCyal10
25
(b)
100
1 0
20
o5
10
Q.
eo
5 4 pmol
- 19kDa
1 0
20
05
10
2
12
(d)
20
8 3 pmol
-20kDa
1 0
16
4
6
8
10 12 14 16
Protein Stquenator Cycle
18
V ( D ) A M R Q N P V X I
20
10
8
12
Elution Time (mm)
Fig. 5. End-stage purification of [3H-CH3]methylated C^-MT peptides by
reversed-phase chromatography (a) peptide CRCl/2, (b) peptide CRC2/2, (c)
peptide CRC3/2, (d) peptide CRC4/2 Calculation of indicated amount and
approximate molecular weight of each peptide is described in the 'Methods' section
Acetonitnle gradient elution conditions (see Table 2) are not indicated, but are
reflected by the A 2)< baseline slope.
enzyme. There are two separate forms of C^-MT in E. coli, an
inducible 39kDa ada gene product (37) and a constitutive 19kDa
ogt gene product (14). Similar separate enzyme forms exist in
B. subtilis of molecular weight 20kDa (constitutive form) and
22kDa (inducible form) (16). The situation regarding multiple
enzyme forms in mammalian tissues is not clear. Subcellular
fractionation of rat liver indicated that the majority of C^-MT
activity is about evenly distributed between nuclear and cytosol
fractions (10,44), which is in contrast to rat spleen, where most
of the enzyme activity is nuclear in location (44). In these studies,
both tissues displayed minor amounts of enzyme activity
associated with mitochondrial and microsomal fractions. More
recently, SDS-PAGE of the methylated enzyme from purified
rat liver mitochondria suggested that this enzyme's molecular
weight was slightly larger than the 23-24 kDa protein detected
in extracts of nuclei purified from the same tissue (45). In
agreement with the findings of the present study was the
observation, mentioned in the latter report, that by the use of
higher resolution SDS-PAGE, several C^-MT proteins were
Fig. 6. Active site region amino acid sequence from human spleen derived [3HCFymethylated peptide CRC2/2 and position of methyl acceptor cysteine (a)
partial sequence of peptide CRC2/2 X, no signal, ( ), uncertainty associated
with amino acid assignment, (( )), very tentative assignment, [ ], assignment
from following radioactivity analysis (b) radioactivity from [3H-CH3]methylated
peptide CRC2/2 associated with - 17% of the sample in each cycle of the protein
sequenator (see accompanying text for further details)
detected in rat liver which displayed slight differences in
molecular size. It is therefore possible that C^-MT may exist in
tissues in distinct molecular forms in a cellular compartmentspecific and even cell type-specific manner.
We describe a procedure for the purification to homogeneity
of fragments of the methylated form of this enzyme. Several
features of our procedure overcome previously encountered
difficulties with regard to C^-MT purification, low abundance
and, in particular, instability. Firstly, extracts of human spleen
were used as an enzyme source. Such extracts contain relatively
high levels of O'-MT activity. Secondly, the methyl group from
C^-MeG becomes covalendy attached to a reactive cysteine
residue in the enzyme, and we exploited this reaction to overcome
the problem of enzyme activity that becomes labile upon
purification, by reacting partially purified C^-MT with substrate
O6-[3H-CH3]methylguanine-DNA to produce a radiolabelled
enzyme whose detection through subsequent purification steps
did not rely upon preservation of enzyme activity. Thirdly, we
chose to purify endogenously produced fragment forms of the
enzyme (under peak I), as they were isolated in purer form than
the presumed larger enzyme (peak II) from which they appeared
to be derived, and to increase the possibility of obtaining highly
purified protein that was not blocked at its AZ-terminus for
downstream amino acid sequence determination. This overall
approach enabled us to obtain homogeneous peptides containing
the C^-MT active site which, as it was anticipated that it might,
facilitated the identification of amino acid sequence with respect
to one such peptide, CRC2/2, as probably belonging to the human
1358 Nucleic Acids Research
(a)
Sequence
source
VD
QG
GA
GO
AS
M
M
N
N
C
RG
RG
GS
K R
AA
P
P
P
D
K
VX AI PC
VP
P C
I S V VP C
L P FVP C
LA
P C
HR V
HRV
HRV
HRV
human epleen
HeLa
£ coll (ogt)
B subt (dat)
£ coll (ads)
Ref
pre»«nl study
1
14
16
37
(b)
a
•A.
a
human spl««n
HeLa
£ coll (ogt)
a
B subt (dat)
a
the methyl acceptor site of C^-MT and, thus, a means for the
detection of any homologous, but distinct, active site region arruno
acid sequences. Such studies with respect to C^-MT of human
spleen are currently in progress in our laboratory.
E coll (ada)
Fig. 7. Comparison of human spleen O*-MT amino acid sequence (peptide
CRC2/2), from the methyl acceptor site cysteine region, with other available
sequences from the same region and their secondary structures (a) uncertainty
of amino acid assignment for the human spleen sequence (see Fig 6) is not
indicated, (b) secondary structure was predicted by the methods of Chou and
Fasman (38) a, a-helix, j3, /3-sheet, turn, /3-turn, r c , random coil The carboxy
end 'turn' was only seen for the human spleen peptide following extension of
the sequence to include the 'HRV part of the conserved pentapeptide sequence
'PCHRV (see Fig 7 (a) above)
enzyme due to its significant homology to corresponding and
similarly homologous sequences from three procaryote forms of
the enzyme.
Interestingly, the pentapeptide sequence -arg-gly-asn-pro-valthat occurs in the human &>-MY sequence is conserved in the
cDNA-denved amino acid sequence from the human DNA
excision repair gene ERCC-1 (46), which corrects the repair
defect of ultraviolet light and mitomycin-C sensitive Chinese
hamster ovary cells belonging to complementation group 1 (47),
but no homology of predicted secondary structure was observed
between these sequences (data not shown).
The strong secondary structural homology seen between
peptide CRC2/2 and the corresponding region of the constitutive
form of C^-MT in E. coli (ogt), and to a slightly lesser extent
with constitutive enzyme in B. subtilis (dat), but least so with
the inducible enzyme in E. coh (ada), suggests the possibility
that peptide CRC2/2 may have originated from a non-inducible
form of C^-MT in human spleen.
Overall, the present purification procedure appears to represent
a useful approach for investigating whether or not separate forms
of Cfi-MT exist in mammalian tissues, by providing a method
for the isolation in pure form of enzyme fragments containing
ACKNOWLEDGEMENTS
We would like to thank Dr. Bryan Smith for affording us protein
sequencing expertise, Rob Nicolas for helpful suggestions in the
protein purification, and Helen Anton for preparing the
manuscript.
This work was supported by grants from the Cancer Research
Campaign (UK) and Medical Research Council (UK).
REFERENCES
1. Tano, K , Shiota, S , Collier, J , Foote, R S and Mitra, S (1990) Proc
Natl Acad Sci USA (in press)
2 NewboW, R F , Warren, W , Medcalf, A S C and Amos, J (1980) Nature
283, 596-598
3 Hams, G , Lawley, P D , Asbery, L J , Chandler, P M and Jones, M G
(1983) Immunology 49, 439-449
4 Zarbl, H, Sukumar, S, Arthur, A V , MarUn-Zanca, D and Barbacid, M
(1985) Nature 315, 382-385
5 Lawley, P.D and Orr, D.J (1970) Chem Biol Interact 2, 154-157
6 Yarosh, D B (1985) Mutat Res 145, 1-16
7 Lindahl, T and Sedgwick, B (1988) Annu Rev Biochem 57, 133-157
8 D'Incalci, M , Citti, L , Taverna, P and Catapano, C V (1988) Cancer
Treatment Reviews 15, 279-292
9 Foote, R S , Mitra, S and Pal, B C (1980) Biochem Biophys Res
Commun 97, 654-659
10 Pegg, A E , Wiest, L , Foote, R S , Mitra, S and Perry, W (1983) J Biol
Chem 258, 2327-2333
11 McCarthy, T V and Lindahl, T (1985) Nucleic Acids Res 13,2683-2698
12 Brent, T P , Dolan, M E , Fraenkel Conrat, H , Hall, J , Karran, P , Laval,
L , Margison, G P , Montesano, R , Pegg, A E , Potter, P M and et al
(1988) Proc Natl Acad Sci USA 85, 1759-1762
13 Dolan, M E , Ophnger, M and Pegg, A E (1988) Mutat Res 193,
131-137
14 Potter, P M , Wilkinson, M C , Fitton, J , Carr, F J , Brennand, J and
Margison, G P (1987) Nucleic Acids Res 15, 9177-9193
15 Rebeck, G W , Coons, S , Carroll, P and Samson, L (1988) Proc Natl
Acad Sci USA 85, 3039-3043
16 Kodama, K , Nakabeppu, Y and Sekiguchi, M (1989) Mutat Res 218,
153-163
17 Morohoshi, F and Munakata, N (1987) J Bactenol 169, 587-592
18 Teo, I A (1987) Mutat Res 183, 123-127
19 Yoshikai, T , Nakabeppu, Y and Sekiguchi, M (1988) J Biol Chem 263,
19174-19180
20 Nakabeppu, Y , Kondo, H , Kawabata, S , Iwanaga, S and Sekiguchi, M
(1985) J Biol Chem 260,7281-7288
21 Bhattacharvya, D , Tano, K , Bunick, GJ., Uberbacher, E C , Behnke, W D
and Mitra, S (1988) Nucleic Acids Res 16, 6397-6410
22. Wilkinson, M.C , Potter, P M , Cawkwell, L , Georgiadis, P , Patel, D ,
Swann, P F and Margison, G P (1989) Nucleic Acids Res 17, 8475-8484
23 Hams, A L , Karran, P and Lindahl, T (1983) Cancer Res 43, 3247-3252
24. Yarosh, D B , Foote, R S , Mitra, S and Day, R S. (1983) Carcinogenesis
4, 199-205.
25. Brent, T P (1986) Cancer Res. 46, 2320-2323
26. Myrnes, B and Wittwer, C U (1988) Eur J Biochem 173, 383-387
27. Hora, J.F., Eastman, A and Bresnick, E (1983) Biochemistry 22,
3759-3763
28 Demple, B , Karran, P , Lindahl, T , Jacobsson, A and Olsson, M (1983)
In Fnedberg, E C and Hanawalt, P C (eds) DNA Repair. A Laboratory
Manual of Research Procedures Marcel Dekker, Inc , New York and Basel,
Vol II, pp 4 1 - 5 2 ,
29. Lawley, P D., Harris, G , Phillips, E , Irving, W , Colaco, C B , Lydyard,
PM and Roitt, I M (1986) Chem Biol. Interact 57, 107-121
30 Renard, A , Verly, W G , Mehta, J R and Ludlum, D B (1983) Eur. J
Biochem 136, 461-467.
31 Schneider, W C (1980) Anal Biochem 103,413-418
32. Lacmmli, U K (1970) Nature 227, 680-685.
Nucleic Acids Research 1359
33 Yarosh, D B , Rice, M , Day, R S , FooCe, R.S and Mitra, S (1984) Mutat
Res 131, 2 7 - 3 6
34. Towbin, H.. Staehehn, T and Gordon, J (1979) Proc Natl. Acad Sci
USA 76, 4350-4354
35 Roberts, P L (1985) Anal Biocnem 147,521-524.
36 Bradford, M M (1976) Anal. Biocnem 72. 248-254
37 Demple, B., Sedgwick, B , Robins, P , Totty, N , Waterfield, M D and
Lindahl, T. (1985) Proc Nat] Acad Sci USA 82, 2688-2692
38 Chou, P Y and Fasman, G D (1978) Adv. Enzymol 47 45-148
39 Mymes, B , Giercksky, K E. and Krokan, H (1982) J. Cell Biocnem. 20,
381-392
40 Yagi, T , Yarosh, D B and Day, R.S (1984) Carcmogenesis 5, 593-600
41 Wiestler, O , Kleihues, P. and Pegg, A E (1984) Carcmogenesis 5, 121-124
42 Fned, V A , Smith, H T , Hildebrandt, E and Werner, K (1987) Proc
Natl Acad. Sci USA 184, 3685-3689
43 Downes, C.S. (1988) Nature 1332, 208-209
44 Jun, G J , Ro, J Y , Kim, M H , Park, G H , Park, W K , Magee, P N
and Kim, S (1986) Biochem Pharmacol 35, 377-384
45 Myers, K A , Saffhill, R and O'Connor, P J (1988) Carcmogenesis 9,
285-292
46 van Duin, M , de Wit, J , Odijk, H., Westcrveld, A., Yasui, A , Koken,
M H.M , Hoeijmakers, J H J and Bootsma, D (1986) Cell 44, 913-923
47 van Duin, M , van den Tol, J , Warmerdam, P , Odijk, H , Meijer, D ,
Westerveld, A , Bootsma, D and Hoeijmakers, J H J. (1988) Nucleic Acids
Res 16, 5305-5322