0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. ..... hypothetical .... types of DNA lesions can be repaired by the human methyl-.
THE JOURNAL OF BIOLOC~CAL CNEMWTRY 0 1990 by The American Society for Biochemistry
Vol. 265, No. 25, Issue of September 5, pp. 14754-14762.1990
and Molecular Biology, Inc.
Purification, Structure, 06-Methylguanine-DNA
Printed in U.S.A.
and Biochemical Properties Methyltransferase”
of Human
(Received
George
Koike&
Hisaji Makiz, of Biochemistry,
From the SDepartment Medical Science, Kyushu
University,
Hiroyuki
TakeyaQ,
Faculty of Medicine Fukuoka 812, Japan
The level of 06-methylguanine-DNA methyltransferase activity in a human cell line carrying a l.lkilobase cDNA fragment was about 50 times higher than that found in ordinary methyltransferase-proficient (Met-+) cell lines (Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Biol. 213, 739-747). Taking advantage of this overproduction, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. A single polypeptide with a molecular weight of approximately 25,000 was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most highly purified preparation. The Stokes radius of 22.5 A and the sedimentation coefficient of 2.0 S were obtained, from which the molecular weight of the native form of the enzyme was calculated to be 19,000. After digestion with lysyl endopeptidase, peptide fragments of the protein were isolated and sequenced. The amino acid sequences of these peptides and the amino acid composition of the protein were in good agreement with those deduced from the nucleotide sequence of the cloned cDNA. The purified enzyme catalyzed transfer of methyl groups from 06-methylguanine and 04-methyfthymine, but not from methylphosphotriesters, of methylated DNA to the enzyme molecule.
Hiroshi
* This work was supported by Grant 61065007 from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March
Hayakawa& and Mutsuo SekiguchiS of Molecular Biology, Graduate School
and the SDepartment
23, 1990)
of
with less methyltransferase activity, in MNU’-administered animals (11, 12). Some human tumor-derived cell lines are hypersensitive to alkylating agents, and these Mer- cells have little or no methyltransferase activity (13-E). When the adu gene of E. coli was introduced into human Mer- cells, the cells acquired resistance to N-methyl-N’-nitro-N-nitrosoguanidine and other alkylating agents (16-19). Attempts have been made to purify @-methylguanine-DNA methyltransferase from human cells (20-24), but no homogeneous enzyme preparation has been obtained, partly due to the low content of the enzyme. Recently we obtained a cell line overproducing methyltransferase after transfection of human Mer- cells with a cDNA expression library derived from methyltransferaseproficient Mer+ human cells (25). The clone carries a cDNA coding for a protein with a significant homology with the bacterial methyitransferases. To ascertain that the cDNA codes for human methyltransferase, we purified the enzyme and compared its amino acid sequence with that deduced from the nucleotide sequence of the cDNA. Here we present evidence that the cDNA indeed codes for the human methyltransferase that repairs 06-methylguanine as well as 04-methylthymine in methylated DNA. EXPERIMENTAL Cell Lines
Alkylating agents are potent carcinogens and induce mutations in various organisms. These effects of alkylating agents appear to be related to formation of various alkylated bases in DNA (1). Among them, 06-methylguanine seems to be most closely linked to induction of mutations and cancers (2, 3). Many organisms carry the enzyme, 06-methylguanine-DNA methyltransferase which catalyzes transfer of methyl groups from 06-methylguanine and other methylated moieties of the DNA to its own molecule, thereby repairing the toxic lesions in a single-step reaction (4). The structure and function of methyltransferase have been studied extensively in Escherichia coli (5-7). Mutations in the ada gene, coding for the 39kDa methyltransferase, lead to an increased susceptibility to alkylating agents, with respect to both mutation induction and cell killing (8-10). Cellular contents of methyltransferase vary with the tissue, and it was pointed out that more tumors are formed in tissues
for publication,
PROCEDURES and Tissue
Culture
HeLa S3 and HeLa MR are methyltransferase-proficient (Mer’) and -deficient (Mer-) strains, respectively (16). Strain X2, which contains a very high level of methyltransferase activity, is the secondary transformant obtained after transfection of HeLa MR cells with a human cDNA library, prepared from Raji (Mer+) cells (25). The cells were maintained in 5% fetal calf serum in Dulbecco’s modified Eagle’s medium containing 100 ,ug/mI streptomycin and 100 units/ml penicillin G. To cultivate the X2 cells, 300 pg/ml hygromycin B was added to ensure retention of the cDNA sequence. Reagents Sources were: N-[3H]methyl-N-nitrosourea (0.5 Ci/mmol) and Enlightning rapid autoradiography enhancer from Du Pont-New Eng land Nuclear; N-[3H]methyl-N-nitrosourea (17.7 Ci/mmol) and “Cradiolabeled protein molecular weight markers from Amersham International (Buckinahamshire): DEAE-Sephacel. Mono S HR5/5, Superose li HR10/30, phosihodiesteraies I and II, poly(dA), poly(dT), and kits of molecular weight markers for gel filtration and sedimentation from Pharmacia LKB Biotechnology Inc. (Uppsala); hygromycin B, BSA, (Fraction V), and calf thy&s DNA (type I) from Sigma; lysyl endopeptidase from Wako Pure Chemical Industries, Ltd. (Osaka); deoxyribonuclease I from Takara Shuzo Co., Ltd. ’ The abbreviations used are: MNU, N-methyl-N-nitrosourea; BSA, bovine serum albumin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high performance liquid chromatography; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SDS, sodium dodecyl sulfate.
14754
Human (Kyoto); (Osaka).
alkaline
phosphatase
(E.
coli)
06-Methylguanine-DNA
from
Toyobo
Co.,
Ltd.
Buffers The following 7.8,0.1 mM EDTA, 50 mM Tris-HCl, 1 mM PMSF, 50 10% glycerol, 0.1 buffer D, 50 mM mM DTT, 1 mM
buffers were used: buffer A, 50 mM Tris-HCl, pH 1 mM DTT, 1 mM PMSF, 70 mM NaCl; buffer B, pH 7.5, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, mM NaCl; buffer C, 50 mM MES . NaOH, pH 6.5, mM EDTA, 2 mM DTT, 1 mM PMSF, 20 mM NaCl; Tris.HCl, pH 7.5, 10% glycerol, 0.1 mM EDTA, 2 PMSF, 100 mM NaCl. Assay
of Methyltransferase
Activity
The methyltransferase activity was measured as described (26), with slight modification. The reaction mixture (100 ~1) contained 70 mM Hepes’KOH (pH 7.8), 1 mM DTT, 5 mM EDTA, 10 pg of BSA, 7.7 pg of [“H]MNU-treated calf thymus DNA (78,000 dpm), and the enzyme. The reaction was carried out by incubating the preparation at 37 “C for 15 min and terminated by adding 500 ~1 of 5% trichloroacetic acid. The mixture was heated at 90 “C for 15 min to hydrolyze the DNA, was placed on ice for 5 min, and then 100 ~1 of BSA (1 mg/ ml) were added. A sample of the mixture was transferred onto a circular Whatman GF/C filter (2.5 cm in diameter) under suction, and the filter was washed 3 times with 5% trichloroacetic acid and once with ethanol. The radioactivity was measured in a liquid scintillation counter. One unit of methyltransferase activity was defined
Amino-terminal Kl Amino acid
Yield
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
GUY Ala GUY Ala Thr Ser GUY Ser Pro Pro Ala GUY Arg Asn
pm01 479 549 473 462 302 132 280 134 213 263 212 190 34 100
Positions
Repetitive yield (%)
of lysyl
K2
no.
Amount of peptide used (pmol)
sequences
AEiy
Yield
423 451 120 146
Purification
Yield
Arg Thr Thr Leu Asp Ser Pro Leu Gb LYS
pm01 143 69 41 142 109 13 41 55 29 10
“a::,’
pm01 122 33 110 46 36
Leu Glu Leu Ser Gly -= Glu Gin Gly Leu Glu Ile LYS
derived
K5 Yield
32 35 24 39 14 59 9
of Human
Methyltransferase
X2 cells were grown to confluence on 150-mm plastic Petri dishes and then were washed with phosphate-buffered saline and scraped into phosphate-buffered saline. The cells were harvested by low speed centrifugation, frozen, and stored at -80 “C until use. The cells (1 X 10”) were suspended in 60 ml of buffer A, homogenized in a Dounce homogenizer (B pestle), and sonicated. All operations were carried out at 4 “C. After centrifugation at 100,000 X g for 10 min at 4 “C, the supernatant was collected (Fraction I, 68 ml). To 66 ml of Fraction I, solid ammonium sulfate was added over a 30-min period to give a final concentration of 0.20 g/ml Fraction I. After leaving to stand at 0 “C for 60 min, the precipitate was removed by centrifugation at 20,000 x g for 40 min at 4 “C. Then another batch of ammonium sulfate was added to the supernatant to give a final concentration of 0.35 g/ml Fraction I, and the precipitate was collected, dissolved in 20 ml of buffer A, and dialyzed overnight against 1 liter of buffer B (Fraction II, 16.3 ml). DEAE-Sephacel Column Chromatography-Fraction II (16 ml) was loaded onto a DEAE-Sephacel column (1.0 x 41 cm) equilibrated with buffer B. Proteins were eluted with 2 column volumes of buffer B at a flow rate of 5 ml/h, and 4.7-ml fractions were collected. Just
K4
Aan!i0
pm01 Leu Leu Gly Lys
as the activity that accepted 1 pmol of methyl group, as described (27). Since the enzyme activity measured by this assay was significantly lower than that obtained with the previous assay procedure (7), the activity was calculated by taking account of this factor, 2.4.
TABLE I endopeptidase peptides
K3
14755
Methyltransferase
AaT$
Yield
Glu Trp Leu Leu Ala His Glu Gly His Arg Leu Gly Lys
pm01 196 322 481 505 293 52 161 144 67 76 214 102 79
from human K6 Aan!o
Yield pm01 10 13 13 13 5 16 15 9 5
Ala Ala Arg Ala Val Gly Gly Ala Met Gly Asn Pro Val Pro Ile Leu Ile Pro
12 16 5 3 6 11 12 6 2
methyltransferase K7
K9
KS
A$zo
Yield
Pro GUY Leu GUY Gly Ser Ser Gly Leu Ala Gly Ala Trp Leu Lys
pm01 277 129 446 139 139 28 65 107 211 258 82 185 77 150 18
An!y
Yield
Phe GUY Glu Val Ile Ser Tyr Gin Gln Leu Ala Ala Leu Ala GUY Asn Pro
pm01 101 141 172 49 122 167 29 173 180 48 240 218 30 189 120 143 21
Aa2$
Yield
Gly Thr Ser Ala Ala Asp Ala Val Glu Val Pro Ala Pro Ala Ala Val Leu Gly GUY Pro Glu Pro Leu Met Gln
pm01 35 19 7 69 69 66 39 20 22 20 29 32 39 32 41 14 19 11 36 19 5 16 4 7 1
TL Ala Trp Leu 194-207
9-18
19-32
650
650
375
188
413
88
525
200
125
(Gly’-Gly”)
ND,,
(Leu4Leu’) 79
(Leu’Leu3) 95
(Glu’*Glu7) 97
(Ala*Alas) 91
(Leu”Leu’) 88
(Leti”Leu’“) 85
(Val”Va116) 94
a -, not identified. b Not determined.
126-144
179-193
108-124
2 3
33-36
99
166-178
1 6
37-66
Human
@-Methylguanine-DNA
after the flow-through fraction, the enzyme activity was eluted, and the most active fractions were pooled and dialyzed against 1 liter of buffer C (Fraction III, 21.5 ml). Mono S Column Chromatography-Fraction III (10 ml) was loaded onto a Mono S HR5/5 column (1 ml) equilibrated with buffer C, and the column was washed with 3 ml of the same buffer. The enzyme activity was eluted with a total of 20 ml of buffer C containing a linear gradient of 20-400 mM NaCl at a flow rate of 0.4 ml/min, and O&ml fractions containing the enzyme activity were pooled (Fraction IV, 1.2 ml). Superose 22 Gel Permeation Chromatography-Fraction IV (1.1 ml) was concentrated to 170 ~1 by a centrifugal microconcentrator, Centricon(M, 10,000 cut off), followed by a gel permeation chromatography on a Superose 12 HR10/30 column (25 ml) equilibrated with buffer D. The column was developed, and 0.25-ml fractions were collected at a flow rate of 0.1 ml/min. The fractions with the highest activity were pooled (Fraction V, 1.25 ml). Lysyl
Endopeptidase
Digestion
and Analyses
of Peptides
The purified human methyltransferase in 50 mM Tris.HCl (pH 9.2) containing 2 M urea was digested with lysyl endopeptidase (an enzyme/substrate ratio of l/50 (w/w)) at 37 “C for 20 h. The digested materials were separated by reversed-phase HPLC with a Cosmosil 3C18 column (4.6 X 100 mm). The isolated peptides were used for amino acid sequence analyses by an Applied Biosystems model 477A gas-phase sequenator, as described by Hewick et al. (28). The phenylthiohydantoin derivatives were analyzed by an Applied Biosystems model 120A phenylthiohydantoin analyzer with an on-line system. Details of determination of the amino acid sequences for an individual peptide are given in Table 1. M
I
II
Ill
IV
V
M
kDa
94 67 43
30
14.4
FIG.
1. SDS-polyacrylamide methyltransferase
gel
electrophoresis of the steps of purification.
hu-
Samples were run in 12.5% polyacrylamide gels containing SDS at 25 mA for 3 h and stained with Coomassie Brilliant Blue. Each lane contained the following materials: I, 50-fig protein of Fraction I; II, 17.4pg protein of Fraction 11; III, 11.9-rg protein of Fraction III; IV, 1.4pg protein of Fraction IV; V, l-pg protein of Fraction V; M, molecular mass markers: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), tu-lactalbumin (14.4 kDa). at various
Determination
of O”-methylguanine-DNA
Analyses
Fraction
units
I. Il. Ill. IV. V.
Crude extract Ammonium sulfate DEAE-Sephacel Mono S Superose 12 “Total methyltransferase
Composition
of Methylated
Bases
Other
Methods
SDS-polyacrylamide gel electrophoresis was essentially as described (32). Protein concentration was determined by the method of Bradford (33) with BSA as a standard. RESULTS
Purification of Human Methyltransferase-The level of methyltransferase activity in the X2 cell line was about 50fold higher than that of normal (Mer+) human cell lines (25). Taking advantage of this overproduction, we purified O’methylguanine-DNA methyltransferase from a crude extract of X2 cells. The extract was processed through ammonium sulfate fractionation and three cycles of chromatography. Purification was followed by SDS-polyacrylamide gel electrophoresis as well as methyltransferase assay (Fig. 1 and Table II). With this procedure, approximately a 550-fold purification was achieved. From 1 x 10” cells, 0.14 mg of the enzyme uds obtained with a yield of 15.8%. As shown in Fig. 2A, an elution profile of the enzyme activity was coincident with that of protein at the final step of purification on Superose 12 gel permeation chromatography. SDS-polyacrylamide gel electrophoresis revealed that the peak fractions contained a single polypeptide with a molecular weight of about 25,000 (Fig. 2B). Judging from these data, the enzyme preparation is apparently homogeneous. Acid Composition
and Sequence of the Purified
amino acid composition of the most purified preparation (Fraction V) was determined, and the data are summarized in Table III. The values obtained from the amino acid analysis matched well the values deduced from the nucleotide sequence of the cloned cDNA (25). Attempts to determine the NHz-terminal sequence of the protein failed, probably due to the presence of a modified amino acid residue at the NHn terminus. Hence, the protein was digested with lysyl endopeptidase, and the resulting peptides were separated by reversed phase HPLC (Fig. 3). Nine peptides were isolated, and their amino acid sequences were determined using an automatic protein sequenator. In Fig. 4, the amino acid sequences thus determined were aligned with the amino acid sequence deduced from the nucleotide seII
methyltransferase
Total activity of methyltransferase”
Acid
[“HIMNU-treated calf thymus DNA or poly(dT) annealed with poly(dA) was incubated with human methyltransferase, and the methylated bases in the polymers were analyzed as described by Lawley and Warren (31) using HPLC with a Waters PBondapak Cl8 column (3.9 X 300 mm). Samples for analyses of OG-methylguanine and 04-methylthymidine were prepared by acid hydrolysis and enzymatic digestion, respectively.
TABLE
Purification
of Amino
The samples were hydrolyzed in uacuo with 5.7 N HCI for 24, 48, and 72 h at 110 “C or with 3 N mercaptoethanesulfonic acid for 24 h at 110 “C by the method of Spackman et al. (29) and Penke et al. (30). Analyses of amino acids were performed with a Hitachi model L-8500 high speed amino acid analyzer.
The Amino Enzyme-The
20.1
man
Methyltransferase
from
a human
Total protein w
31,750 484 30,360 160 20,110 6.67 7,700 0.30 5,020 0.14 activity and protein were calculated for 1 X
cell line, X2,
Specific activity units/mg
65.6 190 3,020 25,700 35,900 lOI cells.
carrying
the cDNA
Purification
Yield
-fold
%
1 2.90 46.0 392 547
100 95.6 63.3 24.3 15.8
Human
06-Methylguanine-DNA
Methyltransferase
14757 TABLE
Amino
acid composition Amino
III
of human methyltransferase
O”-methylguanine-DNA Predicted from nucleotide sequenceh
Analysis”
acid
residues/molecule
M
Fraction 28 29 30 31 32 33 34 35 36 37 36 39 40 41 42 M
kDa
I, .
94 67 Q)
43
m
30
I)
Y
CL
8.9 4 Aspartic acid Asparagine 5 5.8 6 Threonine Serine 11.7 12 20.7 13 Glutamic acid Glutamine 8 18.6 16 Proline Glycine 25.6 27 24.5 25 Alanine Cysteine 4.5d 5 Valine 14.3’ 15 Methionine 3.8 4 Isoleucine 4.7 5 Leucine 23.5 24 Tyrosine 2.9 3 Phenylalanine 5.1 5 Lysine 12.0 12 Histidine 6.6 7 Tryptophan 3.5’ 4 Arginine 6.8 7 Total 207 ” Average values obtained from 24-, 48-, and 72-h hydrolyses with 5.7 N HCl. ’ The data were taken from Hayakawa et al. (25). ’ Extrapolated values to zero time. d Determined as cysteic acid after performic acid oxidation. ’ Taken from 72-h values. ’ Obtained from 24-h hydrolyses with 3 N mercaptoethanesulfonic acid.
14.4
-..
A
Fro. 2. The
final
step
of the
human
methyltransferase
pu-
elution profiles of the activity and the protein from a Superose 12 column. The UV absorbance (-) was measured by a activity Pharmacia UV detector, and the methyltransferase (u) determined as described under “Experimental Procedures.” R, SDS-polyacrylamide gel electrophoresis of the samples from a Superose 12 column. Samples were run in a 12.5% polyacrylamide gel containing SDS at 25 mA for 3 h and stained with Coomassie Brilliant Blue. Each lane contained 15 ~1 each of fractions. Molecular mass markers (M) are as described in the legend to Fig. 1. rification.
A,
quence of the cDNA (25). These sequences, which cover almost half of the entire sequence, matched well those of the hypothetical protein predicted from the cDNA sequence. Thus, it became evident that the cloned cDNA codes for a protein with 06-methylguanine-DNA methyltransferase activity.
Molecular Weight of the Native Enzyme-The molecular weight of the native enzyme was calculated from the Stokes radius and the sedimentation coefficient by the method of Siegel and Monty (34). A plot of the elution position of the enzyme relative t.0 those of reference proteins yielded a Stokes radius of 22.5 A (Fig. 5A). The enzyme was sedimented through a lo-40% (v/v) glycerol gradient, and a sedimentation coefficient of 2.0 S was obtained (Fig. 5B). Using the method of Lee and Timasheff (35), a partial specific volume (v) of the purified enzyme was calculated to be 0.74, based on the amino acid composition of the enzyme shown in Table III. From these data, the molecular weight of the native form of the human methyltransferase was calculated to be 1.9 x 104. The frictional coefficient was calculated from the following equation, f/f0 = a/(3vh4,/4rN)“,
20
0
40
60 Time
60
100
fmin)
B
aS:GTSAADAVEVPAPa*“LGGPEPLIP+TAWL---
FIG. 3. Isolation
and human
amino acid sequencing methyltransferase.
of proteolytic
elution profiles of peptides derived from the human methyltransferase. A sample (200 ~1) of the digest was injected onto a Cosmosil 3C18 column (4.6 x 100 mm) equilibrated with 0.1% trifluoroacetic acid. Peptides were eluted with a linear gradient of O-100% acetonitrile (- - -) in 0.1% trifluoroacetic acid over a period of 100 min at a flow rate of 0.5 ml/ fragments
of the
A,
min at room temperature. B, amino acid sequences of the isolated peptides. NH,-terminal amino acid sequences of peptides (Kl-K9) were determined as described under “Experimental Procedures.” *, not determined.
in which a is Stokes radius, M,, molecular weight, v, partial specific volume, and N, Avogadro’s number; then the value
14758
Human
06-Methylguanine-DNA
Methyltransferase
ATG GAC AAG GAT TGT GAA ATG AAA CGC ACC ACA CTG GAC AGC CCT TTG GGG AAG CTG GAG CTG TCT GGT TGT GAG CAG GGT CTG CAC GAA Met
Asp
Lys
Asp
Cys
Glu
Met
90
Lys K3
K4
ATA AAG CTC CTG GGC AAG GGG ACG TCT GCA GCT GAT GCC GTG GAG GTC CCA GCC CCC GCT GCG GTT CTC GGA GGT CCG GAG CCC CTG ATG 180 TiqpIzzxqGly
ly K2
!Chr Sar
Ala
Na
Asp
Ns
V&l
Glu
Val
Pro
Na
Pro
Na
Ala
Val
bu
Gly
Gly
Pro
Glu
Pro
Lou
&t
60
K9
CAG TGC ACA GCC TGG CTG AAT GCC TAT TTC CAC CAG CCC GAG GCT ATC GAA GAG TTC CCC GTG CCG GCA CTT CAC CAT CCC GTT TTC CAG 270 Gin Cys Zhr Na l'rp &II Am Ala Tyr Phe His Gin Pro Glu Ala Ile Glu Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gin 90
CAA GAG TCG TTC ACC AGA CAG GTG TTA TGG AAG CTG CTG AAG GTT GTG AAA TTC GGA GAA GTG ATT TCT TAC CAG CAA TTA GCA GCC CTG 360 Gin Glu Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys Phe Gly Glu Val 110 Ser Tyr Gin Gin Leu Na Na Lou 120 K0 GCA GGC A&C CCC AAA GCC GCG CGA GCA GTG GGA GGA GCA ATG AGA GGC RAT CCT GTC CCC ATC CTC ATC CCG TGC CAC AGA GTG GTC TGC 450 Cys
His
Arg
Val
Val
Cys
150
K6 AGC AGC GGA GCC GTG GGC AAC TAC TCC GGA GGA CTG GCC GTG AAG GAA TGG CTT CTG GCC CAT GAA GGC CAC CGG TTG GGG AAG CCA GGC 540 Ser Ser Gly Ala Val Gly As,, Tyr Ser Gly Gly Leu Ala Val Lys Glu Trp &II Leu Na Sis Glu Gly Rfs Arg &II Gly Lys 10 Gly 180 K5
K7
TTG GGA GGG AGC TCA GGT CTG GCA GGG GCC TGG CTC AAG GGA GCG GGA GCT ACC TCG GGC TCC CCG CCT GCT GGC CGA AAC TGA 161 Lau Gly Gly Ser Ser Gly i&u Ala Gly Ne !l'rp Leu Lys ly Ns Gly Na Thr Sar Gly Sex Pro Pro Na Gly Arp Aan l **
624 207
Kl
FIG. 4. Amino acid sequence of the human methyltransferase. region of the cDNA (25) and the deduced amino acid seuqence are shown. sequence analyses (Fig. 3) are shown by italic letters in shaded areas. The indicate the isolated peptides (Kl-9).
1.26 was obtained. Thus, the active human methyltransferase seems to be a nearly globular monomer in solution. Methyl Acceptor Capacity of the Enzyme-To show that the methyltransferase protein itself accepts methyl groups from methylated DNA, the purified enzyme was incubated with [3H]MNU-treated DNA and then subjected to SDS-polyacrylamide gel electrophoresis followed by fluorography. As shown in Fig. 6A (lane IV), there is a band corresponding to a 25kDa protein. A band was found at the same position when the reaction was performed with an extract of X2 cells (lane 110. No band was detected with samples of the ordinary Mer’ cells which contain a 50 times lesser amount of the enzyme, and of Mer- cells, virtually defective in the enzyme activity (lanes II and I in Fig. 6A). To detect the methyltransferase of the Mer+ cells, an assay was made with [3H]MNU-treated DNA with higher specific activity. As shown in Fig. 6B, a distinct band was found in the Mer’ cell sample at the same position where the purified enzyme locates (lanes II and 11Z).Even under these conditions the Mer- cell sample shows no band (lane Z). Fig. 7 shows that the purified enzyme rapidly catalyzes transfer of methyl groups from methylated DNA to the enzyme molecule. The reaction was completed within 60 s at 37 “C. The numbers of the transferred methyl groups are roughly proportional to amounts of the protein. Determination
of the Substrate
Specificity
of the Enzyme-
A series of experiments was performed to determine which types of DNA lesions can be repaired by the human methyltransferase. [3H]MNU-treated calf thymus DNA was incubated with various amounts of the purified enzyme, and acid hydrolysates of the DNA samples were analyzed by HPLC (Fig. 8). In the untreated DNA, about 7% of 3H-methylated bases were 06-methylguanine. With increasing amounts of the enzyme, lesser amounts of 06-methylguanine remained in the DNA. After treatment with 6 pmol of the enzyme, 06methylguanine almost completely disappeared from the DNA. Thus, it is evident that the enzyme effectively repairs @-
The nucleotide sequence of the Amino acid residues determined
amino acid sequences enclosed
coding by the by boxes
methylguanine in DNA. Judging from the data shown in Fig. 8, it could be estimated that 1 pmol of the enzyme could accept 0.67 pmol of the methyl group. On the other hand, from the data shown in Fig. 7, it could be estimated that 1 pmol of the enzyme accepts 0.94 pmol of the methyl group. The difference might be due to the methods used for methyltransferase assay. It is likely that one enzyme molecule accepts one methyl group. It has been shown that 04-methylthymine, which is less abundant in methylated DNA, is repaired by the E. coli methyltransferases (36,37). To determine whether the human enzyme also functions to repair 04-methylthymine, we performed the following experiment. Poly(dT) treated with [3H] MNU was annealed with poly(dA), and the polymer was incubated with purified preparations of the E. coli and the human enzymes. The DNA samples were then digested with a mixture of deoxyribonuclease I, phosphodiesterases I and II, and bacterial alkaline phosphatase, and the hydrolysates were analyzed by HPLC. Fig. 9 shows the distribution of the radioactivity of each sample fractionated. There were three peaks, two of which migrated with the authentic markers for methylated nucleosides. The first peak corresponded to Njmethylthymidine and the second one to @-methylthymidine (indicated by an arrow). This second peak reproducibly disappeared when the DNA was treated with either the E. coli or the human enzyme. It appears therefore that the human methyltransferase can repair 04-methylthymine as well as 06methylguanine in DNA. It was noted, however, that the rates of repair of the two methylated bases in the DNA are significantly different (data not shown). Evidence that @-methylthymine can be repaired by the human enzyme has come from another experiment, the result of which is shown in Fig. 6B (lane Iv). The radioactive label was transferred to the methyltransferase molecule when the enzyme was incubated with [3H]MNU-treatedpoly(dT), annealed with poly(dA), in which the radioactivity is present as 04-methylthymine. Thus, it seemed evident that the human enzyme can repair 04-methylthymine.
Human
06-Methylguanine-DNA
14759
Methyltransferase M
A
I
II
Ill
IV
M
kDa
.-$
lb
I
200 92.5
r)
69
kDa 20
30
Stokes
radius
40 200
(A)
92.5 69 46
a
*
30
-a
r)
Fraction FIG. 5. Determination of Stokes radius and sedimentation coefficient of the human methyltransferase. A, a plot for determination of the Stokes radius. Reference proteins (2 mg/ml each) were applied on a Superose 12 column under the same condition as for the purification of human methyltransferase. 0, human OF-methylguanine-DNA methyltransferase; W, ribonuclease A; 0, chymotrypsinogen A; A, ovalbumin; a, BSA. B, a plot for the determination of the sedimentation coefficient. The human methyltransferase (Fraction V, 40 ~1) was mixed with ribonuclease A (1.82 S), chymotrypsinonen A (2.54 S). ovalbumin (3.55 S). and BSA (4.31 S) (2.2 mn/ml each) and centrifuged through a lo-40% glycerol gradient in 50~HIM Tris. HCl, pH 7.5,0.1 mM EDTA, 4 mM DTT, 50 mM NaCl at 55,000 rpm in a Beckman model TLS-55 rotor for 14 h at 4 “C. Samples were collected in 44 fractions from the bottom of the tube. The
third
peak,
which
may
represent
the
radioactivity
derived from methylphosphotriesters, gave characteristic patterns for the two types of enzymes. The peak for the sample treated with the E. coli enzyme was considerably lower than that for the untreated sample, thereby reflecting the fact that the E. coli enzyme (Ada protein) carries a methylphosphotriester-DNA methyltransferase activity (5-7). On the other hand, no decrease in the radioactivity of this peak was observed with the sample treated with the human enzyme. This is consistent with the notion that the human enzyme, which
14.3
FIG. 6. Methyl acceptor capacity of the human methyltransferase. The reaction mixture (COO ~1) contained 70 mM Hepes.KOH (pH 7.8), 1 mM DTT, 5 mM EDTA, 10 pg of BSA, [,‘H]MNU-treated calf thymus DNA, or poly(dT) .poly(dA), and the enzyme. The mixture was incubated at 37 “C for 15 min in the case of calf thymus DNA or 60 min in the case of nolv(dT).nolv(dA). One-half of the mixture was boiled for 90 s in the presence-of 5% 2:mercaptoethanol, 2.3% SDS, 10% glycerol, and 62.5 mM Tris.HCl (pH 6.8) and applied to a 12.5% polyacrylamide gel containing SDS. Electrophoresis was carried out at 25 mA for 3 h, and the gels were fixed with 30% methanol, 10% glacial acetic acid for 30 min, treated with an Enlightning rapid autoradiography enhancer for 15 min, dried, and subjected to fluorography (Fuji x-ray film) at -80 “C. A, the substrate was 15.4 fig of calf thymus DNA (156,000 dpm). Each lane contained the following materials: I, 50-pg protein of HeLa MR (Mer-) extract (Fraction I); II, 50-pg protein of HeLa S3 (Mer+) extract; III, 50-pg protein of X2 extract; IV, 0.3 pg of the purified human methyltransferase (Fraction V); M, molecular mass markers: myosin (200 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), lysozyme (14.3 kDa). B, the following substrates were used: I-III, 5.6 fig of [“HIMNUtreated calf thymus DNA (l,OZO,OOO dpm); IV, 4.4 pg of [“H]MNUtreated poly(dT) annealed with poly(dA) (632,000 dpm). Each lane contained the following materials: I, 50.pg protein of HeLa MR extract; II, 50-pg protein of HeLa S3 extract; III, 0.05-pg protein of the purified human methyltransferase; IV, 0.5 pg of the purified human methyltransferase; M, molecular mas markers as described above.
is significantly smaller than devoid of such an activity. This
notion
the result
was
of which
supported
is shown
is the E. coli Ada protein, by
an
additional
is
experiment,
in Fig. 10. No or only
little
14760
Human
06-Methylguanine-DNA
Methyltransferase
2.a
A
200
0.0 0
10
20
Time
30
(mln)
FIG. 7. Time course of the methyltransferase reaction. The reaction was carried out under the standard assay conditions. Samples were withdrawn from the reaction mixture at the times indicated, and the amounts of methyl groups transferred to the enzyme were determined as described under “Experimental Procedures.” The reaction mixture contained 1 pmol (0) and 2 pmol (0) of the purified enzyme.
transfer of methyl groups occurred with the human enzyme in case of incubation with methylated poly(dA).poly(dT) or methylated poly(dT) .poly(dA) whereas the E. coli Ada protein accepted methyl groups from them as well as from methylated calf thymus DNA. Though the human enzyme can repair 04methylthymine, as shown in Figs. 6B and 9, no band was detected in lane V in Fig. 10; this may be due to the low content of 04-methylthymine in this substrate. DISCUSSION
A cell line with an increased resistance to alkylating agents and an extremely high level of 06-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library prepared from methyltransferase-proficient human Mer’ cells. From the secondary transformant, X2, a cDNA was recovered, which would code for a protein comprising 207 amino acids (25). Since the molecular weight of the hypothetical protein was nearly equal that of the human methyltransferase, it was most likely that the protein coded by the cDNA is the methyltransferase itself. However, there remained the possibility that it encodes a protein that controls expression of the gene for methyltransferase or one regulating the enzyme activity. To obtain a definite conclusion, the amino acid sequence of the human methyltransferase had to be compared with that deduced from the nucleotide sequence of the cDNA. The present study was undertaken to elucidate this question as well as to examine the nature of the methyltransferase reaction. Taking advantage of overproduction of methyltransferase in the transformant cell line, the enzyme was purified to apparent physical homogeneity. The amino acid composition of the purified enzyme preparation matched well one deduced from the nucleotide sequence of the cloned cDNA. The NH,terminal sequence of the protein could not be resolved, probably due to modification of the NH,-terminal amino acid. Thus, we determined part of the internal amino acid sequences and found that the sequence determined, which amounts to almost half of the entire sequence of the protein,
400
200
a 15
Elutlon
tlme
20
(mln)
8. Disappearance of Os-methylguanine from methylated DNA upon incubation with the methyltransferase. t3H] MNU-treated calf thymus DNA (7.7 pg, 78,000 dpm) was incubated with or without methyltransferase (Fraction V) in 100 ~1 of 70 mM Hepes.KOH, pH 7.8, 1 mM DTT, 5 mM EDTA for 15 min at 37 “C. Then 100 ~1 of 1 mg/ml BSA and 400 ~1 of 0.8 M trichloroacetic acid were added, and the DNA was hydrolyzed for 15 min at 90 “C. After centrifugation, 45 ~1 of the acid-soluble hydrolysates were applied to HPLC. The program was a linear gradient of 5-63% methanol in 50 mM HCOONH, (pH 4.60) over a period of 25 min and at a flow rate of 0.8 ml/min at room temperature. The retention times in minutes for the methylated bases were as followed: l-methyladenine, 6.7; 3methyladenine, 8.3; 7-methylguanine, 11.4; @-methylguanine, 14.8. Arrows indicate the radioactivity of 06-methylguanine. A, without enzyme; B, 3 pmol of enzyme; C, 6 pmol of enzyme. FIG,
5
Human
-E 9. E? 2 5 F B n 2
06-Methylguanine-DNA
14761
Methyltransferase
6000
4000
0 12
16
20
12
16
Elutlon
20
12
16
20
time (mln)
FIG. 9. Disappearance of 04-methylthymidine from methylated poly(dT).poly(dA) upon incubation with the methyltransferase. [“HIMNU-treated poly(dT) was annealed with poly(dA), and 7.8 rg of the polymer (64,000) dpm were incubated under the same conditions as described in the legend to Fig. 8. Then the polynucleotide was digested with 500 units of deoxyribonuclease I, 0.5 unit each of phosphodiesterase I and II, and 2 units of alkaline phosphatase (E. co/i) in 250 ~1 of a mixture containing 20 mM MgC12 and 50 mM Tris.HCl (pH 7.6) at 37 “C for 20 h. After removing proteins by precipitation with trichloracetic acid, 150 ~1 of the mixture were applied to HPLC. The set-up was a linear gradient of 30-65% methanol in 50 mM HCOONH, (pH 4.60) over a period of 25 min at a flow rate of 0.5 ml/min at room temperature. The retention times in minutes for the methylated nucleosides were as follows: W-methylthymidine, 14.0; 04-methylthymidine, 16.2. Arrows indicate the radioactivity of O’-methylthymidine. A, without enzyme; B, 15 pmol of the E. coli Ada protein; C, 15 pmol of the human methyltransferase (Fraction V).
IV
v
VI
M
kDa
92.5 69 46
1
30
FIG. 10. Actions of two types of methyltransferases on various substrates. Experimental conditions were as described in the legend to Fig. 6, except that various substrate DNAs were used. 0.2 pg of the E. co/i Ada protein was used for experiments shown in lanes I-III, and 0.3 rg of the human methyltransferase (Fraction V) for those shown in lanes IV-VI. The following substrates were used: I and IV, 15.4 pg of [“HIMNU-treated calf thymus DNA (156,000 dpm); II and V, 15.6 fig of [“HIMNU-treated poly(dT) annealed with poly(dA) (128,000 dpm); III and VI, 13.6 pg of [“HIMNU-treated poly(dA) annealed with poly(dT) (239,000 dpm). Molecular markers (M) are as described in the legend to Fig. 6.
is in accord with that deduced from the cDNA sequence. Taken together, it is now evident that the cDNA encodes the human methyltransferase. While we did our cloning by complementation of the Mer- phenotype with the cDNA (25), two other groups succeeded in cloning of the cDNA, using entirely different procedures. Tano et al. (38) isolated the clone on the basis of its rescue of a methyltransferase-deficient (ada-) E. coli host, and Rydberg et al. (39) cloned it by screening a cDNA library with oligonucleotide probes derived from the active site amino acid sequence of the bovine methyltransferase. The nucleotide sequences of the cDNA, determined independently by the three groups, are practically identical, thus providing further support for the notion that the nucleotide and amino acid sequences presented in Fig. 4 are correct. Partial amino acid sequences of human and other mammalian methyltransferases were recently obtained. Brent et al. (40) and Major et al. (41) isolated SDS-denatured and native [‘HImethyl-accepted human methyltransferase and determined their partial amino acid sequences. The sequences matched well a part of the sequence presented in this work. Methyltransferase enzymes were also purified from rat liver (42) and calf thymus (43), and their partial amino acid sequences were given, which have some homology with those of methyltransferases from other sources. Although attempts have been made to purify the methyltransferase from human cells, there is no documentation of purification of the human enzyme to a homogeneous state. The successful purification in the present study relates to the cellular abundance of the enzyme in the particular cell line, X2. As deduced from the specific activity of the most highly purified preparation, the molecular weight of the enzyme, and the activity in a crude extract, it was estimated that a single X2 cell contains 2.8 x 10’ molecules of methyltransferase.
Human
06-Methylguanine-DNA
Since the level of the enzyme activity in a X2 cell is about 50 times higher than that of a HeLa S3 cell, it can be calculated that a HeLa S3 cell contains about 5 x lo4 enzyme molecules. This value is relatively close to the estimation made for this particular cell line (21, 44). Based on analyses by SDS-polyacrylamide gel electrophoresis followed by fluorography and Western blotting, Gonzaga and Brent (45) estimated the molecular weight of the human methyltransferase to be approximately 25,000. We obtained a similar value by measuring the mobility of the purified enzyme on SDS-polyacrylamide gel electrophoresis. On the other hand, the molecular weight of the native enzyme, calculated from the Stokes radius and the sedimentation coefficient, was 19,000. These values are relatively close to the 21,700 calculated from the nucleotide sequence of the cDNA (25, 38, 39). Taken together, it can be concluded that the enzyme is roughly globular and is present as a monomer in solution. E. coli cells possessat least two types of methyltransferases, a 39-kDa Ada protein (5-7) and a 19-kDa Ogt protein (37). The Ada protein carries two distinct methyltransferase activities, one to transfer methyl groups from methylphosphotriesters and the other to transfer methyl groups from @methylguanine and 04-methylthymine residues of methylated DNA, whereas the Ogt protein carries only the latter activity. With respect to molecular weight, the human enzyme resembles the Ogt protein. Indeed, it was shown, in the present study, that the human enzyme is devoid of the potential to transfer a methyl group from methylphosphotriester in the DNA. There are conflicting reports as to the substrate specificity of mammalian methyltransferases. It has been shown that methyltransferases from human, monkey, and rat can transfer methyl groups from 04-methylthymine (46). On the other hand, there is documentation that the enzyme cannot repair @-methylthymine (47-50). To elucidate the substrate specificity, we performed an HPLC analysis of the DNA treated with the purified human methyltransferase. The result clearly showed that the human enzyme can repair @-methylthymine as well as @-methylguanine in the DNA. In this respect also the human methyltransferase resembles the E. coli Ogt protein (37). extend special thanks to Dr. S. Iwanaga on protein analyses, to Dr. K. Kohda for gifts of various bases and nucleosides, and to M. Ohara for useful com-
Acknowledgments-We
for advice methylated ments.
REFERENCES Strauss, B., Scudiero, D., and Henderson, E. (1975) in Molecular Mechanti:ms for ReDair of DNA (Hanwalt, P. C., and Setlow, R. B., eds) Part A, pp.‘13-26, Plenum Publishing Corp., New York Coulondre, C,, m.rl M;lLr T U (1077, .T MO, Rid 11, 5.77-6WF. Sukumar, S., Notario, V., Martin-Zanca, D., and Barbacid, M. (1983) Nature 306, ,658~661 Lindahl, T., Se ‘dewick, B., Sekiguchi, M., and Nakabeppu, Y. (1988) Annu. Rev. Biochem.-57 ,133-157 Demple, B., Sedgwi ck, B., Robins, P., Totty, N., Waterfield, M. D., and ‘a,,Y
illlll.,‘,
V.
11.
\‘Y
I
*,
V.
1.Z”“.
-II..
LL.,“”
““-
Methyltransferase
Lindahl, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2688-2692 6. Margison, G. P., Cooper, D. P., and Brennand, J. (1985) Nucleic Acids Res. 13,1939-1952 I. Nakabeppu, Y., Kondo, H., Kawabata, S., Iwanaga, S., and Sekiguchi, M. (1985) J. Biol. Chem. 260, 7281-7288 Sedgwick, B., and Robins, P. (1980) Mol. Gcn. Genet. 180,85-90 i: Lemotte, P. K., and Walker, G. C. (1985) J. Bacterial. 161,888-895 10. Takano, Y., and Sekiguchi, M. (1988) J. Mol. Biol. 201, ^^_ ^__K., Nakabeppu, Zbl-Z/l
11. Goth, R., and Rajewsky, M. F. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 639-643 12. Kleihues, P., and Margison, G. P. (1974) J. Natl. Cancer Inst. 63, 18391841 C. H. J., Scud&o, D. A., Meyer, S. A., and 13. Day, R. S., III, Ziolkowski, Mattern, M. R. (1980) Carcinogenesk 1.21-32 14. Yarosh, D. B., Foote, R. S., Mitra, S., and Day, R. S., III (1983) Carcinogenesis 4, 199-205 15. Yagi, T., Yarosh, D. B., and Day, R. S., III (1984) Carcinogenesis 5, 593600 16. Ishisaki, K., Tsujimura,T., Yawata, H., Fujio, C., Nakabeppu, Y., Sekiguchi, M., and Ikenaga, M. (1986) Mutat. Res. 166, 135-141 17. Samson, L., Derfler, B., and Waldstein, E. A. (1986) Proc. Natl. Acad Sci. U. S. A. 83,5607-5610 18. Brennand, J., and Margison, G.P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6292-6296 19. Kataoka, H., Hall, J., and Karran, P. (1986) EMBOJ. 6,3195-3200 20. Mymes, B., Nilsen, I. W., Haugen, A:, and Krokan, H. (1986) in Repair of DNA Lesions Introduced by N-nztroso Compounds (Myrnes, B., and Krokan, H., eds) pp. 112-134, Norwegian University Press, Oslo, Norway T. (1983) Cancer Res. 43, 324721. Harris, A. L., Karran, P., and Lindahl, 3252 22. Brent, T. P. (1984) Cancer Res. 44, 1887-1892 23. Brent, T. P. (1986) Cancer Res. 46.2320-2323 C. U. (1988) Eur. J. Biochem. 173,383-387 24. Mymes, B., and Wittwer, 25. Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Blol. 213,739?“? 26. Mymes, B., Norstrand, K., Giercksky, K., Sjunneskog, C., and Krokan, H. (1984) Carcinogenesrs 5, 1061-1064 27. Demple, B., Jacobsson, A., Olsson, M., Robins, P., and Lindahl, T. (1982) J. Biol. Chem. 257.13776-13780 28. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J. (1981) J. Biol. Chem. 256,7990-7997 29. Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206 30. Penke, B., Ferenczi, R., and Kovacs, K. (1974) Anal. Biochem. 60,45-50 W. (1981) in DNA Repair (Friedberg, E. C., 31. Lawley, P. D., and Warren, and Hanawalt, P. C., eds) Vol. 1, Part A, pp. 129-142, Marcel Dekker, Inc., New York 32. Laemmli, U. K. (1970) Nature 227,680-685 33. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 34. Siegel, “..,. L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346111
sm‘
S. N. (1979) Methods Enzymol. 61,49-57 35. Lee, J. C., and Timasheff, 36. McCarthy, T. V., Karran, P., and Lindahl, T. (1984) EMBO J. 3,545-550 37. Wilkinson, M. C., Potter, P. M., Cawkwell, L., Georgiadis, P., Patek D., Swarm, P. F., and Margison, G. P. (1989) Nucleic Acids Res 17, 84758484 38. Tano, K., Shiota, S., Collier, J., Foote, R. S., and Mitra, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,686-690 B., Spurr, N., and Karran, P. (1990) J. Biol. Chem. 265,956339. Ry$t;;g, 40. Brent, T. P., van Wronski, M., Pegram, C. N., and Bigner, D. D. (1990) Cancer Res. 60,58-61 41. Major, G. N., Gardner, E. J., Came, A. F., and Lawley, P. D. (1990) Nucleic Acids Res. l&1351-1359 M. C., Cooper,,D. P., Southan, C., Potter, P. M., and Margison, 42. Wilkinson, G. P. (1990) Nucleic Actds Res. l&13-16 43. Rydberg, B., Hall, J., and Karran, P. (1990) Nucleic Acids Res. 18, 17-21 44. Foote, R. S., Pal, B. C., and Mitra, S. (1983) Mutat. Res. 119, 221-228 Gonzaga, P. E., and Brent, T. P. (1989) Nucleic Acids Res. 17,6581-6590 2: Becker, R. A., and Montesano, R. (1985) Carcinogenesis 6,313317 41. Dolan, M. E., Scicchitano, D., Singer, B., and Pegg, A. E. (1984) Biochem. Biophys. Res. Commun. 123,324-330 48. Yarosh, D. B., Fornace, A. J., and Day, R. S., III (1985) Carcinogenesis 6, 949-953 6, 1611-1614 49. Dolan, M. E., and Pegg, A. E. (1985) Carcinogenesis 50. Brent, T. P., Dolan, M. E., Fraenkel-Conrat, H., Hall, J., Karran, P., Laval, F., Margison, G. P., Montesano, R., Pegg, A. E., Potter, P. M., Singer, B., Swenberg, J. A., and Yarosh, D. B. (1988) Proc. Natl. Acad. Sci. U. S. A. 86, 1759-1762
