[CANCER RESEARCH 43, 3247-3252,
0008-5472/83/0043-0000$02.00
July 1983]
O6-Methylguanine-DNA
Methyltransferase
of Human Lymphoid Cells:
Structural and Kinetic Properties and Absence in Repair-deficient
Cells
Adrian L. Harris,1 Peter Karran, and Tomas Lindahl2
Imperial Cancer Research Fund, Mill Hill Laboratories, London, NW7 1AD, United Kingdom
ABSTRACT
response to the mutagenic effect of alkylating agents (6). Present
knowledge of the E. coli DNA repair enzyme acting on 06-MeGua
Human lymphoid Å“il lines contain a DNA repair enzyme which
removes the mutagenic alkylation lesion 06-methylguanine from
(10, 12, 21, 22, 31, 35, 37) may be summarized as follows, (a)
The methyl group of an O6-MeGua residue in alkylated DNA is
DNA. The enzyme transfers the methyl group to a protein
cysteine residue, generating S-methylcysteine, and is inactivated
enzymatically transferred to a protein molecule, with the regen
eration of an unsubstituted guanine residue in the DNA. (b) No
release of 06-MeGua occurs in the form of the free base or as
as a consequence of the reaction. Apparently the methylated
enzyme represents a dead-end complex. The transfer reaction
is very rapid and is completed in less than 1 min at 37°, but
methyl group transfer from single-stranded DNA or heavily dam
aged DNA is less efficient. The active methyltransferase and the
methylated protein both have molecular weights of 21,000 to
22,000, as determined by gel filtration. Lymphoid cell lines pro
ficient in repair of O6-methylguanine in vivo, Mex+, contain 10,000
to 25,000 molecules of the methyltransferase per cell. In con
trast, repair-deficient cell lines, Mex~, do not contain detectable
amounts of the enzyme. The latter point was verified by applying
a partial purification procedure for the enzyme to cell-free ex
tracts from two Mex~ cell lines.
INTRODUCTION
The mutagenic effect of simple methylating and ethylating
agents on mammalian cells appears to be largely due to the
formation of miscoding 06-alkylguanine residues in the cellular
DNA (17, 29). A cellular DNA repair activity can to some extent
counteract this mutagenic challenge by rapid removal of O6MeGua3 and 06-ethylguanine from DNA, but the repair function
is of relatively low capacity and is easily saturated on exposure
of cells to alkylating agents (25, 41, 45). It is of interest in this
regard that tumors induced by treatment of animals with simple
alkylating agents arise preferentially in organs or cell populations
with deficient ability to remove 06-alkylguanine from DNA (14,
44). Moreover, while normal human cells have the ability to repair
06-MeGua residues, some human tumors, certain tumor-derived
cell lines, and many lines established from cells transformed with
DNA tumor viruses are deficient in this form of DNA repair. Such
cells, which are anomalously sensitive to alkylating agents, have
been termed Mer (9) or Mex~ (42).
The mechanism of correction of O6-MeGua residues is unu
sual. The biochemistry of the process was first elucidated in
Escherichia coli, in which organism the responsible repair activity
can be induced to high levels and accounts for the adaptive
nucleotide, and there is no generation of apurinic sites or chain
breaks in the alkylated DNA as a consequence of removal of O6MeGua. (c) The receptor moiety is a cysteine residue, and Smethylcysteine is formed in the reaction, (d) The methyltransfer
ase itself is the methyl group acceptor, and each enzyme mole
cule can stoichiometrically remove a single methyl group from
DNA. (e) The transfer reaction is very rapid (completed in 1 sec
at 37°)but occurs by unusual suicide kinetics, reflecting the lack
of regeneration of active enzyme, (f) The methyltransferase is a
monomeric protein of M, 18,000, containing 4 cysteine residues,
only one of which serves as acceptor, (g) The enzyme can
accept alkyl groups from O6-MeGua and the analogous lesion
06-ethylguanine in DNA but not from A/-alkylated purine residues.
In the present work, we have investigated an O6-MeGua-DNA
methyltransferase from human lymphoid cells, in order to deter
mine to what extent the human activity and the E. coli enzyme
are related. Similarities have already been reported from several
laboratories, in that there is stoichiometric transfer of a methyl
or ethyl group in O6-alkylguanine to a protein cysteine residue
by a mammalian methyltransferase, which does not act on A/
methylated purines (3, 26,33, 46). A detailed characterization of
a highly purified enzyme fraction from rat liver has recently been
performed (34). However, no direct information on the molecular
sizes of both the active and methylated protein has been avail
able, and most of the published kinetic data have seemed
anomalously slow in comparison with the results on the E. coli
enzyme. Moreover, the surprising claim has recently been made
that Mer* and Mer cells contain the same amounts of this repair
function (47), although Mer cells do not remove 06-MeGua from
DNA in vivo. If correct, this would be in apparent contrast to the
data obtained in the bacterial system, in which there is a satis
factory agreement between the levels of O6-MeGua-DNA meth
yltransferase and repair capacity for this lesion in vivo between
different strains (38).
MATERIALS AND METHODS
1 Present address: University Department of Radiotherapy and Clinical Oncology,
Newcastle General Hospital, Westgate Road, Newcastle-upon-Tyne, NE4 6BE,
United Kingdom.
* To whom requests for reprints should be addressed.
3The abbreviations used are: O*-MeGua, O8-methylguanine; Mex/Mer, methyl
excision repair phenotype; HPLC, high pressure liquid chromatography; 3-MeAde,
3-methyladenine; 7-MeGua, 7-methylguanine; 3-MeGua, 3-methylguanine.
Received December 6,1982; accepted March 10,1983.
JULY 1983
Cell Lines. The human lymphoblastoid cell lines GM1953, GM0621,
and GM0892A, derived from normal individuals, and GM2246 from a
xeroderma pigmentosum patient, were obtained from the Human Genetic
Mutant Cell Repository, Camden, N. J. The Burkitt lymphoma-derived
cell line Raji was obtained from the American Type Culture Collection,
Rockville, Md. All cells were grown as suspension cultures in Roswell
3247
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1983 American Association for Cancer Research.
A. L Harris et al.
Park Memorial Institute Tissue Culture Medium 1640 supplemented with
10% fetal calf serum. Actively growing cells were harvested by lowspeed centrifugation, washed once with phosphate-buffered saline so
lution, and either used directly for preparation of cell-free extracts or
kept frozen at -80°.
Enzyme Purification. All operations were performed at 2°.Lymphoid
cells (5 x 109 cells) were disrupted in a Potter hand homogenizer in 15
ml of 0.3 M KCI-50 mw Tris-HCI (pH 7.5)-10 ITIMdithiothreitol-1
rriM EDTA-
contained an activity which removed O6-MeGua from DNA, as
measured by the selective disappearance of such residues from
radioactively labeled alkylated DNA. On gel filtration of a crude
cell extract of Raji cells, which exhibited 0.5 unit of methyltrans
ferase activity per mg protein, the enzyme activity eluted as a
single symmetrical peak (Chart 1). By comparison with several
marker proteins, the 06-MeGua-DNA methyltransferase was
cific activity, 1.6 Ci/mmol) as described (21). It contained 1200 cpm per
ng DNA, with about 1 Qua residue in 7000 modified at the O6 position.
found to elute at the position of a globular protein of M, 21,000
±2,000 (S.D.). The 3 most active concentrated fractions were
pooled; they had a specific activity of 8 enzyme units/mg and
contained 5.5 mg protein per ml. The yield of total activity was
about 30%. No further increase in activity was observed on
mixing different column fractions with each other prior to assay
ing, indicating that no separation of the transferase and methyl
acceptor functions had occurred.
The crude cell extract also contained DNA glycosylase activi
ties for the release of uracil, 3-MeAde, 7-MeGua, and 3-MeGua.
The human uracil-DNA glycosylase has a molecular weight of
30,000 (7). The latter 3 activities directed against N-alkylated
purine residues (5,8,24,40) cochromatographed on the Ultrogel
column as one or several proteins of M, 27,000, clearly different
from (and of larger size than) the O6-MeGua-DNA methyltrans
For some experiments, W-methylated purines were removed by hydrol
ysis of the double-stranded DNA substrate at neutral pH prior to use
ferase.
The human O6-MeGua-DNA
(21).
The standard assay for O6-MeGua-DNA methyltransferase measures
the disappearance of O6-MeGua from methylated DNA (21, 22). Reaction
marked heat resistance. Thus, on incubation of the partly purified
enzyme fraction (in the Ultrogel column buffer; Chart 1) at 95°,
mixtures (0.5 ml) consisted of 2 to 6 »galkylated DNA (containing 0.3 to
0.9 pmol O6-MeGua) in 70 mw 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH (pH 7.8)-10 mw dithiothreitol-1 mw EDTA-5% glycerol.
After addition of an O6-MeGua-DNA methyltransferase fraction and in
cubation at 37° (usually for 20 min), the DNA was precipitated, acid
hydrolyzed, and assayed for 06-MeGua content, either by HPLC or after
54% of the methyltransferase activity was retained after 10 min,
and 44% after 20 min. This unusual thermal resistance indicates
that the transferase and methyl acceptor functions reside in the
same heat-stable protein.
Methylated Protein. When the alkylated DNA substrate (pre
viously freed of A/-methylated purines) was incubated with partly
0.5 mM phenylmethylsulfonyl fluoride. Cellular debris was removed by
centrifugation at 10,000 x g for 20 min. One-third of the crude cell
extract was reserved for enzyme and protein assays. Ten ml of the
extract were applied to an Ultrogel AcA-54 (LKB Products, Stockholm,
Sweden) column, 2.6 x 100 cm, equilibrated with 0.5 M NaCI-20 mM TrisHCI {pH 7.5)-10 mw dithiothreitol-1
mw EDTA-0.5 mw phenyl
methylsulfonyl fluoride, and eluted with the same buffer. Individual 5-ml
fractions were collected and concentrated 10-fold in Amicon ultrafiltration
cells equipped with Dia-flo YM2 membranes (Amicon Corp., Lexington,
Mass.). Concentrated protein fractions were assayed for various DNA
glycosylase activities and for O6-MeGua-DNA methyltransferase activity.
Protein concentrations were determined with Coomassie blue (4).
Enzyme Assays. An alkylated DNA substrate was prepared by treat
ment of Micrococcus luteus DNA with A/-[3H]methyl-A/-nitrosourea (spe
methyltransferase
exhibited
a
Dowex-1 chromatography (22). One unit of methyltransferase activity
removes 1 pmol O6-MeGua from DNA under the standard assay condi
10
tions. Experiments on identification of the receptor moiety were per
formed by terminating methyltransferase reactions by digestion with
proteinase K and aminopeptidase M, followed by amino acid analysis as
described (31), except that the aminopeptidase digestion was for 16 hr
(26).
DNA glycosylase activities liberating 3-MeAde, 7-MeGua, and 3MeGua were assayed using [3H]dimethylsulfate-treated
DNA as sub
strate. Assay conditions were the same as those used for the corre
sponding bacterial activities (20) except that reaction mixtures (250 ¿il)
contained 10s cpm and incubations were for 60 min at 37°.The methyl
0-6
'280
>
O
ated purines released into ethanol-soluble form were subsequently quan-
O-4
titated by HPLC analysis.
Molecular Weight of the Methyl Group Receptor. For determination
of the size of the acceptor protein (11), a 4-fold scaled-up complete
assay mixture was supplemented with guanidine hydrochloride, to a final
concentration of 6 M, subsequent to the completion of the methyl transfer
reaction. After 2 hr, the unfolded protein was chromatographed (at 20°)
0-2
on a column (1 x 110 cm) of Sepharose 6B-CL (Pharmacia, Uppsala,
Sweden) equilibrated with 6 M guanidine hydrochloride-1 mw EDTA (pH
5.0). In a separate control experiment, a reaction mixture without added
methyltransferase was chromatographed under identical conditions. The
column had been precalibrated by determinations of the elution volumes
of several reagent proteins of known molecular weight (carbonic anhydrase, chymotrypsinogen,
lysozyme, RNase).
RESULTS
Characterization of the Methyltransferase Activity from Hu
man Lymphoid Cells. Cell-free extracts from the lymphoma line
Raji and the lymphoblastoid cell lines GM1953 and GM2246
3248
200
300
EFFLUENT (ml)
Chart 1. Gel filtration of a cell extract from the human lymphoma line Raji.
Column fractions were assayed for Aj» (
), 3-MeAde-DNA glycosylase (peak
of activity indicated by arrow), and O6-MeGua-DNA methyltransferase activity (•).
The individual fractions were concentrated 10-fold by ultrafiltration prior to the
methyltransferase assays in order to improve accuracy, and 20 ¡aof such concen
trates were added to 0.5 ml standard reaction mixtures. The column had been
precalibrated with myoglobin, dimeric myoglobin, and human uracil-DNA glycosy
lase.
CANCER RESEARCH VOL. 43
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1983 American Association for Cancer Research.
Human 06-MeGua-DNA
purified O6-MeGua-DNA methyltransferase
from Raji and GM
1953 cells, followed by enzymatic hydrolysis of the protein
fraction, the radioactive receptor amino acid residue could be
identified as S-methylcysteine (31). A stoichiometric relationship
between the amount of O6-MeGua removed from DNA and the
amount of S-methylcysteine formed in the methyltransferase
fraction was observed. Thus, in reaction mixtures containing
0.14, 0.28, and 0.56 units O6-MeGua-DNA methyltransferase
from GM 1953 cells (which, according to definition, removed
0.14, 0.28, and 0.56 pmol O6-MeGua from alkylated DNA), 0.12,
0.23, and 0.49 pmol S-methylcysteine, respectively, were re
covered after hydrolysis. These data
enzyme accumulates as a dead-end
tion of activity, as shown previously
ferase (22).
Since transfer of the methyl group
indicate that the methylated
complex with no regenera
for the E. coli methyltrans
from an O6-MeGua residue
to the protein apparently resulted in loss of the enzyme activity,
it was of interest to determine if the molecular size of the
methyltransferase had changed during the process. Therefore,
the methylated protein, carrying radioactive material derived from
06-MeGua in DNA, was denatured in 6 M guanidine hydrochloride. The latter reagent causes complete unfolding of proteins
and allows direct determination of their molecular weights by gel
filtration on a column of cross-linked agarose beads (11). When
a control reaction mixture containing alkylated DNA (not exposed
to methyltransferase but largely freed of A/-methylated purines
by heating at neutral pH) was chromatographed, the DNA ap
peared in the column void volume, with a separate small peak of
low-molecular-weight oligonucleotide material (Chart 2). On gel
filtration of a complete reaction mixture also containing the partly
purified human O6-MeGua-DNA methyltransferase from Raji
cells, the amount of radioactivity associated with the DNA had
diminished, and a new peak of radioactive material appeared
(Chart 2). By comparison with several reference proteins, which
were chromatographed separately, the methyl group acceptor
was determined to have a molecular weight of 22,000 ±1,500.
These data are in good agreement with the determination of the
Methyltransferase
size of the active methyltransferase (Chart 1). We conclude that
there is no detectable change in size of the enzyme on methylation.
Reaction Kinetics. Incubation of alkylated DNA with the partly
purified O6-MeGua-DNA methyltransferase from Raji cells re
sulted in rapid transfer of methyl groups from the O6-MeGua
residues of the DNA substrate. Analysis of DNA hydrolysates by
HPLC showed that the transfer reaction was completed in less
than 1 min at an enzyme concentration sufficient to remove 50%
of the O6-MeGua residues (Chart 3). Since the enzyme is inacti
vated by serving as methyl group acceptor, a fixed amount of
protein removed only a corresponding amount of methyl group
residues from the DNA. Thus, doubling the substrate concentra
tion in a standard reaction mixture did not result in any further
removal of 06-MeGua residues. With enzyme levels sufficient to
remove most or all of the O6-MeGua from the DNA substrate, an
initial rapid phase of transfer was followed by a slower phase,
but the entire reaction was completed in less than 10 min at 37°
(Chart 3). This retardation of the reaction rate may be due to
interference by damage, such as single-strand interruptions and
small gaps, in the double-stranded alkylated DNA substrate.
Single-stranded alkylated DNA was found to be a very poor
substrate for the human O6-MeGua-DNA methyltransferase
(Chart 4). Compared to results obtained with the same DNA in
double-stranded form, the rate of methyl group transfer from O6MeGua residues in single-stranded DNA was at least 100-fold
slower. In this experiment, DNA substrates previously freed of
A/-methylated purines were used. The rate of O6-MeGua removal
from such a double-stranded substrate appeared slightly slower
than for nondepurinated DNA. In addition to the above data
obtained at 37°, the rate of the methyl transfer reaction was
studied at 2 lower temperatures. While the general features of
the reaction kinetics were the same as at 37°,with inactivation
of the enzyme occurring as one consequence, the rate of methyl
group transfer was 12-fold slower at 21 °and 100-fold slower at
5°.
Enzyme Activities in Repair-proficient and Repair-deficient
Cells. The levels of O6-MeGua-DNA methyltransferase activity in
cell extracts of 5 different human lymphoid lines were investi
gated. Three of these cell lines, Raji, GM1953, and GM2246,
have been classified by Sklar and Strauss (42) as proficient in
repair of 06-MeGua, Mex+, while the other 2 lines, GM0621 and
400
a.
<
0-31
300
111
K
<
<
o-il
200
2
<
o
5
100
40
60
80
EFFLUENT (ml)
Chart 2. Gel filtration of the methylated enzyme in its unfolded form. Reaction
mixtures containing DNA with radioactive O6-MeGua residues were incubated with
(•)or without (A) O°-MeGua-DNA methyltransferase and then chromatographed
on a Sepharose 6B CL column equilibrated with 6 M guanidine hydrochloride-1 ITIM
EDTA.
JULY
1983
20
10
TIME
(min)
Chart 3. Kinetics of removal of O*-MeGua from alkylated DNA by the human
methyltransferase. Standard reaction mixtures, each containing 2 fig DNA, were
incubated at 37°with 0.14 units (•)
or 0.28 units (•)
partly purified methyltransfer
ase for various times as indicated. At the end of the reactions, remaining O'-MeGua
in the DNA was analyzed after acidic hydrolysis followed by HPLC.
3249
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1983 American Association for Cancer Research.
A. L Harris et al.
1-0
05
-O
LU
O
•-0-V
/-O-
0-4
OC
z
o---
o
o
E
a
08
D
u
§I
o —'
03
K
111
-
O
0-6
u
111
C
in
0-21-1—L
0 1 2
10
TIME
(min)
Chart 4. Kinetics of removal of O6-MeGua from double-stranded and singlestranded DMA. In this experiment, a DMA substrate largely freed of W-methylated
purines was used. Each reaction mixture contained 3 «¿g
DMA and 0.2 unit
methyltransferase. Prior to the assays at 37°,the DMA had been heated at 100°
for 10 min in 10 nâ„¢Tris MCI, pH 7.5-1 mw EDTA (O) or kept unheated in the same
buffer (•).
GM0892A, were shown to be repair deficient, Mex~. Here,
extracts from the 3 Mex+ cell lines were found to contain O6MeGua-DNA methyltransferase activity, while no such enzyme
activity was detected in the extracts from the Mex~ cells. Data
on 4 of the 5 lines investigated are shown in Chart 5. Raji cells
contained a 2 times higher level of methyltransferase activity (0.5
units/mg) than either GM1953 (0.26 units-mg~1) or GM2246
(0.23 units/mg) cells. A slightly reduced recovery of O6-MeGua
¡nthe standard assay was observed with the Mex~ extracts at
l
04
0-2
0123
PROTEIN
CONCENTRATION
(mg.ml
1)
Chart 5. Capacity for removal of O6-MeGua from alkylated DMA by extracts
from various lymphoid cell lines. Each reaction mixture (0.5 ml) contained 6 ^g
alkylated DNA and different amounts of cell extract as indicated. The sources of
cellular protein were as follows: •,Raji (Mex*); •GM1953 (Mex*); A, GM0892A
(Mex~); T, GM0621 (Mex~); O, controls containing bovine serum albumin instead
of cell extract.
, —, 2 Mex* lines;
, Mex" lines and controls.
high protein concentrations. This may be ascribed to a nonspe
cific effect such as trapping, however, since an identical loss of
radioactive material was seen when the Mex" cell extracts were
06
replaced with bovine serum albumin at equivalent protein con
centrations (Chart 5). As a control, cell extracts from Mex+ and
Mex" lines were assayed separately for uracil-DNA glycosylase.
The preparations contained very similar amounts of this enzyme,
demonstrating that there was no general enzyme deficiency in
the extracts from the 2 Mex~ cell lines.
Since each methyltransferase molecule apparently only acts
once in the standard reaction, the assay is relatively insensitive,
and the enzyme activities in crude extracts of Mex+ cells were
o
o
z
z
0-2
D
O
only detected with certainty at relatively high protein concentra
tions and large reaction volumes (Chart 5). Therefore, extracts
from 2 Mex+ and 2 Mex~ lines were further purified as described
in Chart 1. In each case, the individual column fractions were
assayed for 3-MeAde-DNA glycosylase activity, to allow localiza
tion of the protein fraction within the molecular weight range
18,000 to 25,000. This material was pooled, concentrated by
Ultrafiltration,and assayed for O6-MeGua-DNAmethyltransferase
activity (Chart 6). With these partly purified enzyme fractions,
assays could be performed at lower protein concentrations, and
unspecific losses of O6-MeGua were no longer a problem, as
judged from the data obtained with the Mex~ material (Chart 6).
The results confirmed and extended those obtained with the
crude cell extracts. Thus, while the 2 Mex+ lines contained readily
detectable levels of methyltransferase, the Mex" lines did not
show any significant enzyme activity. From the data in Chart 6,
we estimate that a Mex" line such as GM0892A contains less
than 2% of the O6-MeGua-DNAmethyltransferase activity of the
Mex+GM 1953 line.
3250
PROTEIN CONCENTRATION
(mg.ml ')
Chart 6. Capacity for removal of 06-MeGua from alkylated DNA by partly purified
enzyme fractions from lymphoid cells. Each enzyme preparation contained a
concentrate of the protein fractions of molecular weight similar to the C^-MeGuaDNA methyltransferase. For further details, see text. Symbols as in Chart 5.
In further experiments, cell extracts from Mex" lines were
added to extracts from Mex+ lines. The material from the Mex"
cells neither inhibited nor stimulated the O6-MeGua-DNAmeth
yltransferase activity of the Mex+ extracts. These data show that
the Mex" cell extracts did not contain an inhibitor of the enzyme.
Instead, such cells appear to have shut off production of the
methyltransferase. These latter results are ¡ngood agreement
with the in vivo studies by Ayres et al. (1) on the relative DNA
repair capacities of hybrids of fused Mex+ and Mex" cells.
In E. coli, induction of the O6-MeGua-DNAmethyltransferase
by alkylating agents is coordinately regulated with the induction
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1983 American Association for Cancer Research.
VOL. 43
Human O6-MeGua-DNA Methyltransferase
Table 1
DNA glycosylase activities against N-methylated purines in extracts from various
human lymphoid cell lines
Enzyme activities were determined at several different protein concentrations
within the linear range of the assays, and liberated methylated purines were
separated by HPLC prior to radioactivity determinations.
with
regard to
O'MeGua re
lineRaji
Cell
1.1
0.11
0.7
0.13
1.12.53-MeGua0.09
0.07
0.087-MeGua0.50
transferase. These data are in good agreement with experiments
in vivo (39, 41). In this context, the strongly reduced enzyme
activity on alkylated single-stranded DNA (Chart 4) is of interest,
since it suggests that the critical O6-MeGua residues in the
vicinity of DNA replication forks would be inefficiently repaired.
From the specific radioactivities and O6-MeGua contents of
(units)83-MeAde1.7
glycosylase activities
moval
(42)Mex+
Mex*
GM1953
Mex*
GM2246
MexGM0621
Wlex-DNA
GM0892APhenotype
observed is limited by the level of the O6-MeGua-DNA methyl
0.41
0.64
0.51
0.68
Units are expressed as pmol base excised per mg protein per hr.
the alkylated DNA substrates used, the protein concentrations
of the active cell extracts, and the assumption that the human
activity shares the property with the E. coli enzyme of removing
only a single methyl group from alkylated DNA, we estimate that
the human lymphoid cell lines GM1953, GM2246, and Raji con
tain 10,000 to 25,000 06-MeGua-DNA methyltransferase mole
cules per cell. Similar levels of O6-MeGua-DNA methyltransferase
of a DMA glycosylase, termed 3MeAde-DNA glycosylase II, which
can excise 3-methyladenine and 3-MeGua from DMA (20). The
increased ability to remove A/-3-methylated purines from DNA
causes increased resistance to the cytotoxic action of methylating agents. Since Mex~ cells are more sensitive than Mex+ cells
are found in extracts of human fibroblasts (19). These numerical
estimates are essentially the same as those of Sklar ef a/. (41)
but seem lower than other published reports. In general, human
cells appear somewhat more repair-proficient for O6-MeGua than
to killing by such agents (16,42), the levels of the 3-MeAde- and
3-MeGua-DNA glycosylase activities as well as the 7-MeGua-
rodent cells (33). For comparison, bacteria such as E. coli and
Salmonella typhimurium have an 800 to 900 times smaller ge
nome and contain 20 to 30 O6-MeGua-DNA methyltransferase
DNA glycosylase activity (40) were determined in the crude cell
extracts of the various lymphoid cell lines. As shown in Table 1,
there were no significant differences between Mex+ and Mex~
cells with regard to the relative amounts of these enzyme activ
ities. (It is not known if these human DNA glycosylase activities
against W-methylated purines reside in one enzyme of relatively
broad substrate specificity or in several enzymes). Thus, the
absence of O6-MeGua-DNA methyltransferase activity in the
Mex" lymphoid cell lines was not accompanied by a similar
deficiency in DNA glycosylase activity against W-methylated pu
rines. It is presently unclear if 06-MeGua or another alkylation
product is the main cytotoxic lesion in human cells (2, 8,16, 43),
but the anomalous sensitivity of Mer and Mex" cells to alkylating
agents does not appear to reflect deficient repair of the major Nalkylated purines.
DISCUSSION
The basic features of the reaction between the human O6MeGua-DNA methyltransferase and alkylated DNA seem very
similar to those of the extensively investigated E. coli system.
Thus, transfer of a methyl group to a cysteine residue of the
protein occurs in a rapid reaction, with accumulation of the
methylated enzyme as a dead-end product. Several research
groups have already obtained similar results with the correspond
ing enzyme from rodent cells (3, 26, 33). Minor differences
between the bacterial and mammalian proteins include the
slightly larger size of the mammalian enzyme, Mr 21,000 to
22,000 observed here for the human activity and just over 20,000
for the rat liver enzyme (34), in comparison with the E. coli
protein of M, 18,000 (10). Furthermore, in parallel kinetic experi
ments, the human enzyme investigated here seemed more sen
sitive to inhibition by strand interruptions and apurinic sites and
also seemed less active at decreased temperatures than the
bacterial enzyme (22). Similar observations have been made
previously for the rat liver and mouse liver methyltransferases
(3,34). The apparently slow reaction kinetics reported by several
groups (3,26,46) may be due to high inhibitory DNA and protein
concentrations in reaction mixtures. Thus, under optimal reaction
conditions, the human enzyme completes repair of O6-MeGua
residues in less than 1 min (Chart 3), and the amount of repair
JULY 1983
molecules per cell (10, 15, 27), so human lymphoid cells and
these gram-negative bacteria contain very similar amounts of the
activity per unit of DNA. The E. coli B/r enzyme can be induced
to over 100-fold higher levels by exposure of cells to simple
alkylating agents (6). Such an adaptive response does not occur
in mammalian cells, which appear to be basically noninducible
(19, 23). A moderate (about 5-fold) increase in O6-MeGua-DNA
methyltransferase in rat hepatocytes in response to exposure to
hepatotoxic agents or surgical damage appears to be a different
phenomenon from the adaptive response of E. coli, especially
since no similar induction has been observed in other rodent
species (28,30, 32). It is noteworthy that the adaptive response
to alkylating agents characteristic of E. coli is by no means
universal and does not even occur in all related bacterial species.
Thus, S. typhimurium only exhibits a low level of constitutively
expressed O8-MeGua-DNA methyltransferase activity (15) and
seems similar to mammalian cells in this respect.
The DNA repair-deficient Mex~ lymphoid cell lines investigated
here did not contain detectable amounts of O6-MeGua-DNA
methyltransferase activity, even after attempts at enrichment by
a partial purification procedure (Charts 5 and 6). Thus, there is a
clear difference in enzyme levels between Mex+ and Mex~ cells.
Similar results have recently been independently obtained by
Sklar et al. (43) and by Foote et al. (13) for the equivalent Mer+
and Mer phenotypes. These data do not agree with a recent
claim by Waldstein ef al. (47) that Mer+ and Mer cell extracts
contain similar amounts of O6-MeGua-DNA methyltransferase
activity. The experimental discrepancies cannot be ascribed to
differences between various repair-deficient cell lines, because
the Mer lines characterized by Foote ef al. (13) were identical
with some of those studied by Waldstein ef al. (47). While we
cannot explain the anomalous results of the latter group, it is
noteworthy that these workers only used a single, relatively low
amount of protein in their assays on crude cell extracts. As seen
here in the left part of Chart 5, it was difficult to detect consistent
differences between Mex+ and Mex~ cell extracts under such
experimental conditions, while significant nonspecific losses of
06-MeGua occurred (as demonstrated here with a control con
taining bovine serum albumin instead of enzyme). Similar obser
vations could perhaps have been misinterpreted as reflecting O63251
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1983 American Association for Cancer Research.
A. L Harris et al.
MeGua-DNA methyltransferase activity.
In conclusion, Mex" (or Mer) cells and cell extracts, with the
obvious exception of ' 'leaky" strains, appear to differ from Mex+/
Mer* cells in being totally deficient in 06-MeGua-DNA methyl
transferase. A suitable bacterial model for this situation is not
presently available, because mutants with an inactive structural
gene for the enzyme have not been found, although noninducible
E. coli strains containing the constitutive level of enzyme activity
are available (18, 27, 36). Since Mex cells presumably would
be particularly vulnerable to simple alkylating agents, the con
venient means of characterization offered by direct enzyme
assays with cell-free extracts might be useful for their identifi
cation.
20.
21.
22.
23.
24.
25.
26.
ACKNOWLEDGMENTS
We thank lain Goldsmith and Shirley Williams for performing large numbers of
HPLC experiments and Dr. Sankar Mitra, Dr. Anthony Pegg, and Dr. Bernard
Strauss for preprints of their papers.
REFERENCES
1. Ayres, K., Sklar, R., Larson, K., Undgren, V., and Strauss, B. Regulation of
the capacity for O'-methylguanine removal from DNA in human lymphoblastoid
cell lines studied by cell hybridization. Mol. Cell Biol., 2: 904-913,1982.
2. Baker, R. M., Van Voorhis, W. C., and Spencer, L. A. HeLa cell variants that
differ in sensitivity to monofunctional alkylating agents, with independence of
cytotoxic and mutagenic responses. Proc. Nati. Acad. Sei. U. S. A., 76: 52495253, 1979.
3. Bogden, J. M., Eastman, A., and Bresnick, E. A system in mouse liver for the
repair of O'-methylguanine lesions in methylated DNA. Nucleic Acids Res., 9:
3089-3103,1981.
4. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem., 72. 248-254,1976.
5. Brent, T. P. Partial purification and characterization of a human 3-methyladenine-DNA glycosylase. Biochemistry, 18:911-916,1979.
6. Caims, J., Robins, P., Sedgwick, B., and Talmud, P. Inducible repair of alkylated
DNA. Prog. Nucleic Acid Res. Mol. Biol., 26. 237-244, 1981.
7. Caradonna, S. J., and Cheng, Y. C. Uracil-DNA glycosylase: purification and
properties of this enzyme isolated from blast cells of acute myetocytic leukemia
patients. J. Bra). Chem., 255. 2293-2300, 1980.
8. Cathcart, R., and Gokjthwait, D. A. Enzymatic excision of 3-methyladenine
and 7-methylguanine by a rat liver nuclear fraction. Biochemistry, 20: 273-
cytotoxic effects of W-methyl-N-nitrosourea is apparently absent in normal
human fibroblasts. Biochimie (Paris), 64: 717-721, 1982.
Karran, P., Hjelmgren, T., and LJndahl, T. Induction of a DNA glycosylase for
W-methyiated purines is part of the adaptive response to alkylating agents.
Nature (Lond.), 296: 770-773,1982.
Karran, P., Lindahl, T., and Griffin, B. Adaptive response to alkylating agents
involves alteration in situ of O'-methylguanine residues in DNA. Nature (Lond.),
280: 76-77, 1979.
LJndahl, T., Demple, B., and Robins, P. Suicide inactivation of the £.coli O'methylguanine-DNA methyltransferase. EMBO. J., 7: 1359-1363,1982.
Margison, G. P. Effect of pretreatment of rats with MNU on the repair of O'methylguanine in liver DNA. Carcinogenesis (Lond.), 2: 431-434, 1981.
Margison, G. P., and Pegg, A. E. Enzymatic release of 7-methylguanine from
methylated DNA by rodent liver extracts. Proc. Nati. Acad. Sei. U. S. A., 78:
861-865,1981.
Medcalf, A. S. C., and Lawley, P. D. Time course of O'-methylguanine removal
from DNA of MNU-treated human fibroblasts. Nature (Lond.), 289: 796-798,
1981.
Mehta, J. R., Ludlum, D. B., Renard, A., and Verly, W. Repair of O'-ethylgua
nine in DNA by a chromatin fraction from rat liver: transfer of the ethyl group
to an acceptor protein. Proc. Nati. Acad. Sei. U. S. A., 78: 6766-6770, 1981.
27. Mitra, S., Pal, B. C., and Foote, R. S. O'-Methylguanine-DNA methyltransferase
in wild-type and ada mutants of E. coli. J. Bacterio)., 752: 534-537,1982.
28. Montesano, R., Brésil,H., Planche-Martel, G., Margison, G. P., and Pegg, A.
E. Effect of chronic treatment of rats with dimethylnitrosamine on the removal
of O'-methylguanine from DNA. Cancer Res., 40: 452-458, 1980.
29. Newbold, R. F., Warren, W., Medcalf, A. S. C., and Amos, J. Mutagenicity of
carcinogenic methylating agents is associated with a specific DNA modification.
Nature (Lond.), 283: 596-599, 1980.
30. O'Connor, P. J., Chu, Y. H., Cooper, D. P., Maru, G. B., Smith, R. A., and
Margison, G. P. Species differences in the inducibility of hepatic O'-alkykjuanine repair in rodents. Biochimie (Paris), 64: 769-774, 1982.
31. Olsson, M., and Lindahl, T. Repair of alkylated DNA in E. coli: methyl group
transfer from O'-methylguanine to a protein cysteine residue. J. Biol. Chem.,
255:10569-10571,1980.
32. Pegg, A. E., and Perry, W. Stimulation of transfer of methyl groups from O'methylguanine in DNA to protein by rat liver extracts in response to hepatotoxins. Carcinogenesis (Lond.), 2:1195-1200,1981.
33. Pegg, A. E., Roberfroid, M., Von Bahr, C.. Foote, R. S., Mitra, S., Brésil,H.,
Ukhachev, A., and Montesano, R. Removal of O'-methylguanine from DNA by
human liver fractions. Proc. Nati. Acad. Sei. U. S. A., 79: 5162-5165,1982.
34. Pegg, A. E., Wiest, L., Foote, R. S., Mitra, S., and Perry, W. Purification and
properties of O'-methylguanine-DNA
transmethylase from rat liver. J. Biol.
Chem., 258: 2327-2333,1983.
35. Robins, P., and Caims, J. Quantitation of the adaptive response to alkylating
agents. Nature (Lond.), 280: 74-76, 1979.
36. Sedgwick, B. Genetic mapping of ada and ade mutations affecting the adaptive
response of E. coli to alkylating agents. J. Bacteriol., 750: 984-988,1982.
37. Sedgwick, B., and Lindahl, T. A common mechanism for repair of O'-methyl
guanine and O'-ethylguanine in DNA. J. Mol. Biol., 754. 169-175,1982.
280,1981.
9. Day, R. S., Ziolkowski, C. H. J., Scudiere, D. A., Meyer, S. A., Lubiniecki, A.
S., Girardi, A. J., Galloway, S. M., and Bynum, G. D. Defective repair of
alkylated DNA by human tumour and SV40-transformed human cell strains.
Nature (Lond.), 288: 724-727,1980.
10. Demple, B., Jacobsson, A., Olsson, M., Robins, P., and LJndahl, T. Repair of
alkylated DNA in E. coli: physical properties of O*-methylguanine-DNA meth
yltransferase. J. Biol. Chem., 257: 13776-13780, 1982.
11. Fish, W. W., Mann, K. G., and Tanford, C. The estimation of polypeptide chain
molecular weights by gel filtration in 6 M guanidine hydrochloride. J. Biol.
Chem., 244: 4989-4994, 1969.
12. Foote, R. S., Mitra, S., and Pal, B. C. Demethylation of O'-methylguanine in a
38. Sedgwick, B., and Robins, P. Isolation of mutants of E. coli with increased
resistance to alkylating agents: mutants deficient in thiols and mutants consti
tutive for the adaptive response. Mol. Gen. Genet., 780: 85-90, 1980.
39. Shiloh, Y., and Becker, Y. Kinetics of O'-methylguanine repair in human normal
synthetic DNA polymer by an inducible activity in E. coli. Biochem. Biophys.
Res. Commun., 97: 654-659, 1980.
13. Foote, R. S., Pal, B. C., and Mitra, S. Quantitation of O'-methylguanine-DNA
42.
methyltransferase in HeLa cells. Mutât.Res., in press, 1983.
14. Goth, R., and Rajewsky, M. F. Persistence of O'-ethylguanine in rat brain DNA:
43.
15.
16.
17.
18.
19.
correlation with nervous system specific carcinogenesis by ethylnitrosourea.
Proc. Nati. Acad. Sei. U. S. A., 71: 639-643,1974.
Guttenplan, J. B., and Milstein, S. Resistance of Salmonella typhimurium TA
1535 to O*-guanine methylation and mutagenesis induced by low doses of Wmethyl-A/'-nitro-N-nitrosoguanidine:
an apparent constitutive repair activity.
Carcinogenesis (Lond.), 3: 327-331,1982.
Harris, G., Lawley, P. D. and Olsen, I. Mode of action of methylating carcino
gens: comparative studies of murine and human cells. Carcinogenesis (Lond.),
2:403-411,
1981.
Heffch, R. H., Beranek, D. T., Kode«,R. L, and Morris, S. M. Induction of
mutations and sister-chromatid exchanges in Chinese hamster ovary cells by
ethylating agents. Mutât.Res., 706:147-161,1982.
Jeggo, P. Isolation and characterization of E. coli K-12 mutants unable to
induce the adaptive response to simple alkylating agents. J. Bacteriol., 739:
783-791,1979.
Karran, P., Arieti, C. F., and Broughton, B. C. An adaptive response to the
3252
and ataxia telangiectasia cell lines and correlation of repair capacity with
cellular sensitivity to methylating agents. Cancer Res., 41: 5114-5120,1981.
40. Singer, B., and Brent, T. P. Human lymphoblasts contain DNA glycosylase
activity excising N-3 and N-7 methyl and ethyl purines but not O*-alkylguanines
or 1-alkyladenines. Proc. Nati. Acad. Sei. U. S. A., 78: 856-860,1981.
41. Sklar, R., Brady, K., and Strauss, B. Limited capacity for the removal of O'-
44.
45.
methylguanine and its regeneration in a human lymphoma line. Carcinogenesis
(Lond.), 2: 1293-1298, 1981.
Sklar, R., and Strauss, B. Removal of O'-methylguanine from DNA of normal
and xeroderma pigmentosum-derived lymphoblastoid cell lines. Nature (Lond.),
2S9. 417-420, 1981.
Sklar, R., Strauss, B., Ayres, K., Larson, K., Lindgren, V., and Sagher, D.
Cellular aspects of DNA repair. Proceedings of the 13th International Cancer
Congress. New York: Alan R. LJss, Inc., in press, 1983.
Swenberg, J. A., Bedell, M. A., Billings, K. C., Umbenhauer, D. R., and Pegg,
A. E. Cell-specific differences in O"-alkylguanine DNA repair activity during
continuous exposure to carcinogen. Proc. Nati. Acad. Sei. U. S. A., 79: 54995502,1982.
Teo, I. A., and Karran, P. Excision of O'-methylguanine from DNA by human
fibroblasts determined by a sensitive competition method. Carcinogenesis
(Lond.), 3. 923-928, 1982.
46. WakJstein, E. A., Cao, E. H., Miller, M. E., Cronkite, E. P., and Setlow, R. B.
Extracts of chronic lymphocytic leukemia lymphocytes have a high level of
DNA repair activity for O'-methylguanine. Proc. Nati. Acad. Sei. U. S. A., 79:
4786-4790, 1982.
47. Waldstein, E. A., Cao, E. H., and Setlow, R. B. Adaptive resynthesis of 0emethylguanine-accepting protein can explain the differences between mam
malian cells proficient and deficient in methyl excision repair. Proc. Nati. Acad.
Sei. U. S. A., 79: 5117-5121,1982.
CANCER
RESEARCH
VOL. 43
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1983 American Association for Cancer Research.
O6-Methylguanine-DNA Methyltransferase of Human Lymphoid
Cells: Structural and Kinetic Properties and Absence in
Repair-deficient Cells
Adrian L. Harris, Peter Karran and Tomas Lindahl
Cancer Res 1983;43:3247-3252.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/43/7/3247
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1983 American Association for Cancer Research.