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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 46, Issue of November 12, pp.

33002–33010, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Recombinant Human DNA (Cytosine-5) Methyltransferase


I. EXPRESSION, PURIFICATION, AND COMPARISON OF DE NOVO AND MAINTENANCE METHYLATION*

(Received for publication, May 12, 1999, and in revised form, August 16, 1999)

Sriharsa Pradhan, Albino Bacolla‡, Robert D. Wells‡, and Richard J. Roberts§


From New England Biolabs, Beverly, Massachusetts 01915 and the ‡Center for Genome Research, Institute of Biosciences
and Technology, Texas A & M University, Texas Medical Center, Houston, Texas 77030-3303

A method is described to express and purify human methyl group donor S-adenosyl-L-methionine (AdoMet)1 to the
DNA (cytosine-5) methyltransferase (human DNMT1) us- active site of the enzyme. DNA (cytosine-5) methyltransferases
ing a protein splicing (intein) fusion partner in a bacu- (m5C MTase) introduce a methyl group onto carbon 5 of the
lovirus expression vector. The system produces ~1 mg of target cytosine through a covalent intermediate between the
intact recombinant enzyme >95% pure per 1.5 × 109 protein and the target cytosine (9). During this process, the
insect cells. The protein lacks any affinity tag and is cytosine is flipped 180° out of the DNA backbone into an active
identical to the native enzyme except for the two C- site pocket of the enzyme (10). After completion of the methyl
terminal amino acids, proline and glycine, that were transfer reaction, the products, methylated DNA and S-adeno-
substituted for lysine and aspartic acid for optimal syl-L-homocysteine (AdoHcy), are released. Previous studies on
cleavage from the intein affinity tag. Human DNMT1 the mechanism of methylation were mainly limited to prokary-
was used for steady-state kinetic analysis with poly(dI- otic m5C MTases although some limited kinetic studies have

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dC)·poly(dI-dC) and unmethylated and hemimethylated also been reported with mouse and human DNMT1 (11, 12).
36- and 75-mer oligonucleotides. The turnover number
Eukaryotic m5C MTases have been cloned from various or-
(kcat) was 131–237 h—1 on poly(dI-dC)·poly(dI-dC), 1.2–2.3
h—1 on unmethylated DNA, and 8.3– 49 h—1 on hemimeth- ganisms such as mouse (13), human (14), Xenopus (15), sea
ylated DNA. The Michaelis constants for DNA (Km CG
) and urchin
human (16),
DNMT1 chicken (17), localized
has been Arabidopsis (18),
to the and pea (19).
chromosomal siteThe
19
AdoMet
S-adenosyl-
from 0.33–1.32
L -methionine (AdoMet)
and 2.6 –7.2 (K ) ranged p13.2-p13.3 by fluorescence in situ hybridization (14). The en-
m
µM, respectively, whereas the
ratio of kcat/KCG ranged from 3.9 to 44 (237–336 for zyme consists of a large N-terminal region and a smaller C-
poly(dI-dC) m 6 —1 —1 terminal region linked by a run of Gly-Lys dipeptide repeats
·poly(dI-dC)) × 10 M h . The preference of
the enzyme for hemimethylated, over unmethylated, (14). The large N-terminal domain is unique to the eukaryotic
DNA was 7–21-fold. The values of kcat on hemimethyl- m5C MTases. The smaller C-terminal domain has strong ho-
ated DNAs showed a 2–3-fold difference, depending mology with prokaryotic m5C MTases and contains the ele-
upon which strand was pre-methylated. Furthermore, ments necessary for catalysis (20). The function of the N ter-
human DNMT1 formed covalent complexes with sub- minus is poorly understood, and there is little sequence
strates containing 5-fluoro-CNG, indicating that sub- homology between plant and animal enzymes. Sequences
strate specificity extended beyond the canonical CG within this domain have been implicated in Zn 2 binding (21),
dinucleotide. These results show that, in addition to interaction with proliferating cell nuclear antigen (22), and
maintenance methylation, human DNMT1 may also targeting to replication foci (8). The full-length mouse m5C
carry out de novo and non-CG methyltransferase activ-
MTase is about 1700 amino acids in length and has a molecular
ities in vivo.
mass of 183.5 kDa (23).
Prokaryotic m5C MTases are usually part of restriction-mod-
ification systems and rarely discriminate between unmethyl-
Methylated cytosine is found in the genome of organisms
ated or hemimethylated DNA substrates. In contrast, mamma-
ranging from prokaryotes to mammals (1). Methylation of DNA
lian m5C MTases show a much higher reaction velocity on a
in eukaryotes is implicated in various biological and develop-
hemimethylated DNA substrate (maintenance methylation),
mental processes, such as gene regulation (2), DNA replication
the product of semiconservative DNA replication, than on an
(3), genomic imprinting (4), embryonic development (5), carci-
unmethylated DNA substrate (de novo methylation). Methyla-
nogenesis (6), and genetic diseases (7). The bulk of the methy-
tion of single-stranded DNA by the mammalian m 5C MTase is
lation takes place during DNA replication in the S-phase of the
strongly stimulated by the presence of nearby 5-methylcy-
cell cycle (8). The maintenance methylation ensures the prop-
tosines (24). This rate of methylation was comparable with that
agation of tissue-specific methylation patterns established dur-
of hemimethylated DNA (24). It has been demonstrated that
ing mammalian development. The methyl transfer reaction the human enzyme can also methylate non-B-DNA structures
proceeds via nonspecific binding of the enzyme to DNA, recog- such as hairpins and mismatches (12). Recently, a family of
nition of the specific DNA target site, and recruitment of the
mammalian m5C MTases has been identified and hypothesized
to serve as de novo methyltransferases (25).
* This work was supported by National Institutes of Health Grants
GM46127 (to R. J. R.), NS37554, and GM52982 (to R. D. W.) and by the
Robert A. Welch Foundation (to R. D. W.). The costs of publication of 1
The abbreviations used are: AdoMet, S-adenosyl-L-methionine;
this article were defrayed in part by the payment of page charges. This AdoHcy, S-adenosyl-L-homocysteine; m5C MTase, DNA (cytosine-5)
article must therefore be hereby marked “advertisement” in accordance methyltransferases; kbp, kilobase pair; AcNPV, Autographa californica
with 18 U.S.C. Section 1734 solely to indicate this fact. nuclear polyhedrosis virus; DTT, dithiothreitol; PAGE, polyacrylamide
§ To whom correspondence should be addressed: New England Bio- gel electrophoresis; bp, base pair(s); FdC, 5-fluoro-2'-deoxycytidine;
labs, 32 Tozer Rd., Beverly, MA 01915. Tel.: 978-927-3382; Fax: 978- SNRPN, small nuclear riboprotein-associated peptide N; FMR, fragile
921-527; E-mail: roberts@neb.com. X mental retardation.

33002 This paper is available on line at http://www.jbc.org


Methylation by Recombinant Human DNA Methyltransferase 33003
The diverse role played by DNA methylation in mammals were checked for methyltransferase activity and fusion protein expres-
has motivated several groups to express mammalian m 5C sion by Western blot analysis using anti-intein antibody (New England
Biolabs). For routine protein expression, SF9 cells were grown in spin-
MTases in Escherichia coli (26), baculovirus (23, 27), and mam-
ner culture flasks. SF9 cells at a density of 1.4 × 106 ml—1 were infected
malian (COS) cells (28). In the E. coli and COS cell expression at a multiplicity of infection between 5 and 7. The cells were kept at
systems, the amount of protein produced was low, whereas 27 °C at 60 rpm and harvested 48 h post-infection after a final wash
protein expression was high using the baculovirus system. with phosphate-buffered saline.
Insect cells are rich in proteases, and long purification proto- Recombinant DNA (Cytosine-5) Methyltransferase Purification—For
cols often result in partial proteolytic degradation of proteins. protein purification, infected cells (5.6 × 108) were resuspended in 15
To overcome the problem of degradation, we have constructed ml of buffer M (50 mM Tris-HCl, pH 7.4, 1 mM Na2EDTA, protease
inhibitor mixture containing 4-(2-aminoethyl)benzenesulfonyl fluoride,
and expressed the human DNMT1 as a fusion protein with a pepstatin A, E64, bestatin, leupeptin and aprotinin (Sigma), 0.2% (v/v)
Saccharomyces cerevisiae VMA1 intein gene (29). The C termi- per ml of cell extract, 7 µg/ml phenylmethylsulfonyl fluoride, and 500
nus of the human DNMT1 was fused to the N terminus of the mM NaCl). The cell suspension was sonicated on ice for 30 s using a
intein. A small chitin-binding domain from Bacillus circulans model W-225R (Heatsystem-Ultrasonics) sonicator in pulsed mode at
has been added to the C terminus of the intein for affinity 50% duty cycle. The extract was incubated on ice for 30 min with
purification. Thus, the three-part fused human DNMT1 protein occasional shaking and was centrifuged at 11,000 × g for 30 min. If the
supernatant remained cloudy, then one more centrifugation step was
can be immobilized using chitin beads as an affinity matrix. 2
included. Purification from larger numbers (>1 × 109) of cells required
Addition of dithiothreitol (DTT) at low temperature initiates
an initial 40 –70% ammonium sulfate fractionation. This supernatant
cleavage on the column between the intein and the recombi- was loaded on a chitin bead column (New England Biolabs) equilibrated
nant human DNMT1.2 Whereas the intein and chitin-binding with buffer M at 0.4 ml/min. The nonspecifically bound proteins were
domain remain attached to the column, the purified recombi- removed by washing with 30-column volumes of buffer M. On-column
nant enzyme is eluted. This purified recombinant human cleavage of the target protein was initiated by passing 2-column vol-
DNMT1 is identical with the native enzyme except for the two umes of buffer M supplemented with 50 m M DTT. The column was
closed, and the target protein was cleaved from the intein chitin binding
C-terminal amino acids. In this report, we have studied its

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domain by overnight incubation at 4 °C. Human DNMT1 was eluted
steady-state kinetic parameters on double-stranded, unmethy- with buffer M and dialyzed against buffer M supplemented with 0.2%
lated, and hemimethylated DNA substrates, which are repre- (v/v) protease inhibitor mixture (Sigma), 7 µg/ml phenylmethylsulfonyl
sentative of human genes. We also assayed methylation of fluoride, 100 mM NaCl, 50% glycerol (v/v), and 1 mM DTT. The purified
non-CG sequences by human DNMT1. protein was stored at —20 °C. The purity of the protein was checked by
SDS-PAGE (4 –20% Tris/glycine/SDS gradient gel). Purified protein
EXPERIMENTAL PROCEDURES was quantitated using the Bradford assay with bovine serum albumin
Human DNA (Cytosine-5) Methyltransferase Transfer Vector—Hu- as standard.
man DNMT1 expression constructs were derived from pBKSHMT5.0 DNA (Cytosine-5) Methyltransferase Assay and Data Analysis—m5C
(b) (gift of Prof. S. Baylin, The Johns Hopkins University). This plasmid MTase assays were carried out at 37 °C, for 30 min in duplicate with a
had the full-length human DNMT1 cDNA based on the previously total volume of 25 µl of reaction mix. A typical reaction contained S-
published sequence (14). A polymerase chain reaction of the 3' end of adenosyl-L- [methyl-3H]methionine (AdoMet) (specific activity 15 Ci/
the cDNA was used to incorporate an SmaI restriction endonuclease mmol, Amersham Pharmacia Biotech), substrate DNA, and enzyme in
site in place of the stop codon of the cDNA and to provide the optimal assay buffer (50 mM Tris-HCl, pH 7.8, 1 mM Na2EDTA, pH 8.0, 1 mM
amino acids for protein cleavage (sense primer, 5'-GGAATTCCATAT- DTT, 7 µg/ml phenylmethylsulfonyl fluoride, 5% glycerol, and 100
GCATGACAGGAAGAACGGCCGCAGC-3' and antisense primer, 5'-T- µg/ml bovine serum albumin). For kinetic analysis, the CG or CI con-
GACCCGGGAGCAGCTTCCTCCTCCTTTATTTTAGC-3'). The restric- centrations were calculated from either substrate oligonucleotides or
tion sites are underlined. This change causes the C-terminal amino poly(dI-dC)·poly(dI-dC). One nM double-stranded oligonucleotide with 1
acids lysine and aspartic acid to become proline and glycine. Substitu- CG site is 2 n M CG. For poly(dI-dC)·poly(dI-dC), an average length of
tion of these two amino acids at the C terminus of the mammalian 7000 bp was used to determine the concentration of CI. For double-
DNMT1 had no effect on the methyl transfer reaction as compared with reciprocal plots, the concentrations of CG or CI and AdoMet were
the native sequence enzyme as judged by methylase assay (data not varied. The MTase reactions were stopped by transferring the reaction
shown). This polymerase chain reaction product of about 500 bp was tubes to an ethanol/dry ice bath and were processed by spotting the
digested with NdeI and SmaI and ligated to a baculovirus vector reaction mix on DE81 paper circles (Whatman). These circles were
pVIC12 to give pAVICHMT-4. This construct contains the 3' end of the washed sequentially with 4 × 1 ml of cold 0.2 M ammonium bicarbonate,
human DNMT1 cDNA in frame with the intein chitin-binding domain of 4 × 1 ml of Milli Q water, and 4 × 1 ml of ethanol using a manifold
the transfer vector pVIC1.2 pAVICHMT-4 was digested with NruI and (Millipore) connected to a vacuum pump. The filters were dried; 3 ml of
EagI and a 4.1-kbp EcoRV-EagI fragment from pBKSHMT5.0(b) was Opti-fluor (Packard) was added to each, and tritium incorporation was
ligated to give the functional human DNMT1 transfer vector pVICHMT measured. To calculate counting efficiency, tritiated plasmid DNA
(Fig. 1). The ligated junctions in the final pVICHMT construct were (pUC DNA methylated with tritiated AdoMet using M.SssI) was either
verified by DNA sequencing. spotted on DE81 paper, processed, and counted or directly counted with
Insect Cell Culture, Viral Transfection, and Recombinant Protein Opti-fluor. The efficiency of [3H]DNA measurement was ~55%, and all
Expression—A pupal ovarian cell line (SF9) from the worm Spodoptera calculations were corrected accordingly. Data obtained were plotted by
frugiperda was used for co-transfection and expression of the human regression analysis using the GraphPad PRISM program (GraphPad
DNMT1 as described previously (23) with the following modifications. Software Inc.) or SigmaPlot (SPSS Inc., Chicago, IL). The equations
SF9 cells were maintained as a suspension culture in TNM-FH media used for fitting the data points and to obtain the kinetic constants, Vmax,
(JRH Biosciences) supplemented with fetal calf serum 10% (v/v) and an KmCG
, KAdoMet , are reported elsewhere (32). These figures represent es-
antibiotic/antimycotic solution at a final concentration of 5 units of m
timated values.
penicillin, 50 µg of streptomycin, and 0.125 µg of amphotericin B ml—1 Southern Blot Analysis—Genomic DNA was isolated from SF9 cells
at 27 °C on a Bellco steering platform at 70 rpm. Co-transfection of a 48 h post-infection with recombinant virus using the Easy DNA Kit
monolayer of SF9 insect cells was carried out using BaculoGold DNA, a (Invitrogen). The purified DNA was digested either by AatII, BsiEI, or
modified, linearized Autographa californica nuclear polyhedrosis virus SwaI, and the digested DNA fragments were separated on a 1% agarose
(AcNPV) DNA (PharMingen) and the transfer vector pVICHMT. Four
gel in TBE buffer gel. The DNA in the gel was blotted on to Hybond-N+
days after transfection, the supernatant was screened for recombinant
and probed with a random-primed human DNMT1 clone. The blot was
baculovirus using the agarose overlay technique (31). Individual
washed as described (33) and autoradiographed.
plaques were purified and amplified 2 to 3 times in order to reach a viral
Western Blot Analysis—Cell extracts were mixed with SDS loading
titer above 2 × 108 ml—1. Recombinant clones from individual plaque
dye with 2 µM Tris-(2-carboxyethyl) phosphine in place of DTT, boiled at
isolates were used for test expression by infecting 1.5 × 106 SF9 cells in
95 °C for 5 min, and loaded on a 4 –20% Tris/glycine/SDS gradient gel.
a 60-mm Petri dish. Forty-eight hours post-infection the cell extracts
Tris-(2-carboxyethyl) phosphine is a strong reducing agent and does not
take part in the cleavage process (34). The proteins were blotted on
2
M. E. Scott, S. Pradhan, and M. Q. Xu, submitted for publication. an Immobilin-P membrane and probed with an anti-intein antibody
33004 Methylation by Recombinant Human DNA Methyltransferase
(New England Biolabs) followed by chemiluminescent detection of the
human DNMT1.
5-Fluoro-2'-deoxycytidine Assay—Duplex oligonucleotides containing
5-fluoro-2'-deoxycytidine (FdC) were 32P-end-labeled using polynucle-
otide kinase and [μ-32P]ATP. Human DNMT1 (10 n M) was used with 5
nM fluorocytosine-containing oligonucleotide duplex in the presence of
50 µM cold AdoMet in 1× buffer M at 37 °C for 30 min. The reaction was
stopped by adding SDS gel loading dye (one-third of the reaction vol-
ume) and boiling the sample at 100 °C for 5 min. The boiled protein/
DNA mixtures were loaded and resolved on a 4 –20% Tris/glycine/SDS
gradient gel. The gel was dried and autoradiographed.
Oligonucleotides—Poly(dI-dC)·poly(dI-dC), average length 7000
bp, was obtained from Amersham Pharmacia Biotech. The following
olig- onucleotides were synthesized at New England Biolabs. For the
SNRPN exon-1: UMUS, d(CAGAGTGGAGCGGCCGCCGGAGATGCC-
TGACGCATCTGTCTGAGGAGCGGTCAGTGACGCGATGGAGCGGG-
CAAG); MUS, d(CAGAGTGGAGMGGCMGCMGGAGATGCCTGAMG-
CATCTGTCTGAGGAGMGGTCAGTGAMGMGATGGAGMGGGCAA-
G); UMLS, d(CTTGCCCGCTCCATCGCGTCACTGACCGCTCCTCAG-
ACAGATGCGTCAGGCATCTCCGGCGGCCGCTCCACTCTG); MLS,
d(CTTGCCMGCTCCATMGMGTCACTGACMGCTCCTCAGACAGAT-
GMGTCAGGCATCTCMGGMGGCMGCTCCACTCTG). For the FMR-1
locus: UMUS, d(CGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCG-
GCGG); MUS, d(MGGMGGMGGMGGMGGMGGMGGMGGMGGMG-
GMGGMGG); UMLS, d(CCGCCGCCGCCGCCGCCGCCGCCGCCGC-
CGCCGCCG); MLS, d(CMGCMGCMGCMGCMGCMGCMGCMGCMG-
CMGCMGCMG). For the FC oligo duplexes: FS (FG), d(GGAGATGT-

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CFGCAACFGGATACFGAAGGAACCTGC); MLS, d(GCAGGTTCCTT-
MGGTATCMGGTTGMGGACATCTCC); FS (FCG), d(GGAGATGTFC-
GCAAFCGGATAFCGAAGGAACCTGC); FS (FWG), d(AGATGTGCFT-
GCFAGFTGAGFAGGATC); MLS (MWG), d(GATCMTGCTMAGMTG-
GMAGGCACATCT). The following abbreviations were used for
oligonucleotide duplexes: UMUS, unmethylated upper strand; MUS,
methylated upper strand; UMLS, unmethylated lower strand; MLS,
methylated lower strand; FS, fluorocytosine strand; W is either A or T;
M is 5-methylcytosine; and F is 5-fluoro-2'-deoxycytidine. Duplexes
were prepared by annealing equimolar amounts of appropriate single
strands. Complementary oligonucleotides were incubated in annealing
buffer (50 mM Tris-HCl, pH 7.4, 1 mM Na2EDTA, and 100 mM NaCl) at
95 °C for 5 min followed by 65 °C for 15 min and 37 °C for 15 min. For
the oligonucleotides corresponding to the CGG·CCG repeat of the
FMR-1 locus, the annealing step was followed by cleavage with S1
nuclease. This additional step was included to eliminate any single-
stranded nucleotide that resulted from imperfect annealing between FIG. 1. Expression of recombinant human DNA (cytosine-5)
the two complementary 36-mers. PAGE analyses of the annealed du- methyltransferase. A, diagram representing the human DNMT1 ex-
plexes showed that ~90% of the products had the expected length of 36 pression construct pVICHMT and its method of integration into the
bp, whereas ~10% were one repeat shorter (11 repeats, 33 bp in length). AcNPV genome. pVICHMT is shown schematically. A 4.85-kbp cDNA
The full-length duplexes were purified on a 15% polyacrylamide gel, containing human DNMT1, indicated by the gray shading, was fused to
dissolved in TE, and stored at —20 °C. the N-terminal of the VMA1 intein-chitin binding domain, as shown by
the hatched line. Two flanking black arrows corresponding to AcNPV
RESULTS open reading frame (ORF) 603 and 1629 are also indicated. The Amp
and ori loci are indicated with black shading. The recipient AcNPV
Expression and Purification of the Biologically Active Human genome has deletions of the following sequences, C-terminal ORF1629,
DNA (Cytosine-5) Methyltransferase—Co-transfection of the polyhedrin promoter (pPH), and the polyhedrin open reading frame.
human DNMT1 transfer vector, pVICHMT, with linear AcNPV The N-terminal intein cleavage site APG/C, is also shown. The regions
DNA (BaculoGold DNA, PharMingen) resulted in homologous available for homologous recombination are indicated. B, a schematic of
recombination of the polyhedrin promoter and the DNMT1 VICHMT8, a recombinant virus following homologous recombination.
The polyhedrin promoter lies upstream of the DNMT1-intein-CBD fu-
cDNA carried by the construct (Fig. 1, A and B). Only the sion genes. The full-length cDNA was used as a probe to identify the
recombinant viruses are viable. The transfection supernatant correct homologous recombination. The restriction sites are indicated as
was harvested and used for purification of several single A (AatII), S (SwaI), and B (BsiEI). C, Southern blot analysis of the DNA
plaques. Recombinant viral DNA from each of the plaques was from the recombinant clone VICHMT8. The DNA band released by
SwaI contains the whole cDNA plus the intein-CBD, which is about 10
isolated and checked for correct recombination of the human kbp. The other two enzymes release the expected fragments of 3.5 and
DNMT1-intein-chitin binding domain construct using restric- 8.0 kbp for AatII and 2.5 and 6.5 kbp for BstEI. Minor bands represent
tion enzyme digestion and Southern blotting. The probe used defective viral DNAs resulting from illegitimate recombination (30). D,
was the full-length human DNMT1 cDNA. As expected, an time course of human DNMT1 expression using VICHMT8. Infected
cell extracts were analyzed on an SDS gel, blotted, and probed with
SwaI digest gave an ~10-kbp band, whereas AatII and BsiEI anti-intein antibody. A 235-kDa band appears in all lanes. The day of
gave two bands each (Fig. 1C). Several clones had double ho- cell harvest is indicated at the top of each lane. Control lanes with
mologous integration with the human DNMT1 coding sequence uninfected SF9 cell extract are labeled C. M indicates the biotinylated
lying downstream of the polyhedrin promoter, and one, protein molecular weight markers. BSA, bovine serum albumin.
VICHMT8, was used for all further expression and purification
studies. Two different insect cell lines, SF9 and High Five been reported to express between 5- and 10-fold higher protein
(Trichoplusia ni), were used for the evaluation of protein levels. levels than SF9 cells (35), but for human DNMT1 the protein
Different multiplicities of infection were used, and the extracts expression did not improve (data not shown). All further exper-
were evaluated at 48 h post-infection for DNMT1 expression iments used the SF9 cell line for expression. To follow expres-
using poly(dI-dC)·poly(dI-dC) as substrate. High Five cells sion and find optimal conditions for production, extracts of the
have
Methylation by Recombinant Human DNA Methyltransferase 33005

FIG. 2. Single step purification of recombinant human DNA (cytosine-


5) methyltransferase. Protein samples were resolved on a 4 –20%
Tris/glycine gel and visualized by Coomassie staining. M in kDa
(molecular mass markers), E (cell extract), A (ammonium sulfate frac-
tion), F (flow-through), and W (wash) are as indicated on the top of the
gel. Column fractions are numbered. Arrows indicate the approximate
location of the purified human DNMT1 and chitinase, respectively.

human DNMT1 clones were assayed by Western blots using


anti-intein antibody. The optimal expression of the enzyme was
48 h post-infection (Fig. 1D), and the human DNMT1-specific
band starts decreasing after 54 h post-infection infection (data
not shown).

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Mouse DNMT1 is highly unstable and susceptible to prote-
olysis (13). The protease-susceptible domains in mouse and FIG. 3. Linearity of the methyl transfer reaction as a function
human DNMT1 are similar (14). To minimize degradation, all of time and enzyme concentration. A, time course of methylation
catalyzed by recombinant human DNA (cytosine-5) methyltransferase.
steps of the purification were carried out at 4 °C using buffers Reactions (500 µl) containing 1 nM human DNMT1, poly(dI-dC)·poly(dI-
containing a mixture of protease inhibitors supplemented with dC), and AdoMet were incubated at 37 °C. 20 µl of reaction mix in
additional E64, a strong inhibitor of the cysteine protease, duplicate were spotted onto DE81 at fixed time intervals and processed
which is abundant in insect cells. During sonication, the pres- as described under “Experimental Procedures.” 3H incorporation in the
DNA was measured. The mean values of two measurements are plotted.
ence of 500 mM NaCl ensures that most of the enzyme, which is Filled circles, 100 nM CI and 1 µM AdoMet; open circles, 1 µM CI and 10
normally tightly bound to DNA, remains soluble (36). Most of µM AdoMet B, linearity of the methyl transfer reaction as a function of
the enzyme was bound to the chitin-bead affinity matrix. Over- enzyme concentration. Duplicate reactions (25 µl) contained poly(dI-
night incubation at 4 °C with 50 m M DTT induced cleavage on dC)·poly(dI-dC) (1 µM CI), 10 µM AdoMet, and various human DNMT1
the column and released most (>90%) of the full-length recom- concentrations (0.4, 0.8, 1.2, 2.0, 3.0, and 4.0 n M). The filled circles
depict the mean values of [3H]methyl group incorporation.
binant human DNMT1 (Fig. 2). This method yielded protein
that was at least 95% pure as determined by SDS-PAGE on a
4 –20% Tris/glycine gradient gel and Coomassie staining ent CI (DNA) concentrations, with poly(dI-dC)·poly(dI-dC) hav-
(Fig. 2). The dialyzed protein was stored at —20 °C for 4 weeks ing an average length of 7000 bp. With this procedure the
with little loss of activity. However, significant loss of activity reciprocal of the amount of [3H]CH3 transferred to the DNA in
appeared upon prolonged storage (>6 weeks) at either —20 or 1 min by 1 nM DNMT1 (1/v) is plotted as a function of 1/sub-
—80 °C. strate concentration (either 1/CI or 1/AdoMet) in the presence
Steady-state Kinetic Properties of Recombinant Human DNA of fixed concentrations of the co-substrate. For bireactant en-
(Cytosine-5) Methyltransferase on Poly(dI-dC)·Poly(dI-dC) Sub- zymes, which use two substrates and give two products such as
strate—Historically, poly(dI-dC)·poly(dI-dC) has been used to DNMT1, these double-reciprocal plots usually yield linear re-
define the biochemical properties of mammalian m 5C MTases gressions, and their slopes and intercepts are then replotted
(36, 37). Each molecule of poly(dI-dC)·poly(dI-dC) provides a to derive all the kinetic constants. However, for poly(dI-
large number of potential (CI) dinucleotide sites for methyla- dC)·poly(dI-dC), both double-reciprocal plots, i.e. those with
tion. It has also been claimed that this polymer can undergo 1/CI as the variable substrate (Fig. 4A) as well as those with
methylation at rates comparable to a hemimethylated DNA 1/AdoMet as the variable substrate (Fig. 4B), were not linear.
substrate (38). Thus, we used this substrate to define the bio- In particular, the increases in 1/v at low 1/CI (i.e. at high DNA
chemical properties of the recombinant human DNMT1. The concentration) indicated that the DNA substrate inhibited the
time course of methylation for poly(dI-dC)·poly(dI-dC) was ex- reaction, especially in the presence of low amounts of AdoMet
amined at constant DNA and enzyme concentrations. The re- (Fig. 4A). We conclude that poly(dI-dC)·poly(dI-dC) acts both as
action was found to be linear for the first 30 min (Fig. 3A), a substrate and as an inhibitor of the methyl transfer reaction
during which time each enzyme molecule undergoes several with human DNMT1. The regressions from the plots of 1/v
rounds of catalysis. This is evident from the fact that 1 nmol of versus 1/AdoMet (Fig. 4B) were concave, suggesting some pos-
human DNMT1 incorporated several nanomoles of methyl itive cooperativity due to enzyme activation at high AdoMet
groups onto the substrate DNA (Fig. 3, A and B). Similar concentrations. Overall, these data indicate that the methyl
reactions were carried out with fixed concentrations of DNA transfer reaction with poly(dI-dC)·poly(dI-dC) by human
and AdoMet but variable amounts of enzyme. The rate of meth- DNMT1 does not follow simple kinetics.
ylation was linear at enzyme concentrations between 1 and 4 Since the derivation of the Michaelis constants and Vmax
nM (Fig. 3B). The resulting curves (Fig. 3, A and B) were used requires an estimate of the slopes and intercepts from the lines
to determine the optimal enzyme and substrate concentrations in Fig. 4, the two data points at low 1/CI that deviated from
under which linear methylation reaction was obtained. Based linearity at the four lowest AdoMet concentrations (Fig. 4A)
on the above observations, a series of double-reciprocal plots were excluded from the regressions (32, 39). Fig. 5, A and B,
was made at six different AdoMet concentrations or six differ- shows that, with the exception of 1 µM AdoMet, such slopes and
33006 Methylation by Recombinant Human DNA Methyltransferase

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FIG. 4. Double-reciprocal plots for poly(dI-dC)·poly(dI-dC). A,
the AdoMet concentration was fixed at 1.0 µM (filled diamonds), 1.7 µM
(filled squares), 2.6 µM (filled triangles), 5.0 µM (filled circles), 10 µM
(open circles), and 20 µM (open squares). 1/v is plotted against 1/CI.
Linear regression used 4 sets of data points (0.1, 0.12, 0.17, and 0.25 µM
CI) because substrate inhibition was observed at higher CI concentra-
tions (0.5 and 1 µM). The values of the slope and intercept were used to
replot the data and obtain the kinetic constants. B, the DNA (CI)
concentration was fixed at 0.1 µM (open diamonds), 0.12 µM (filled
diamonds), 0.17 µM (open squares), 0.25 µM (filled squares), 0.5 µM
(open circles), and 1.0. µM (filled circles). 1/v is plotted against 1/AdoMet
using a non-linear regression. The intersects on the y axis and the
slopes were replotted to obtain the second set of kinetic constants. The
average values of the kinetic constants are shown in Table I.

intercepts varied linearly with respect to 1/AdoMet concentra-


tion, which enabled all the kinetic constants to be evaluated
(32). The intercept values from Fig. 4B were also replotted (Fig.
5C) and indicated that Vmax(app) varied linearly with the DNA
FIG. 5. Intercept and slope replots of the methylation rate in
at infinite AdoMet concentrations. Furthermore, the 1/Vmax poly(dI-dC)·poly(dI-dC). Replots of the slopes and y axis intercepts
obtained from these data (y axis intercept of Fig. 5C) agreed are from Fig. 4. A and B represent the slopes versus 1/AdoMet and
closely with that estimated from Fig. 5B (y axis intercept), 1/Vmax(app) versus 1/AdoMet from the fixed AdoMet data in Fig. 4A.
giving confidence in the method used. Due to their curvature, Crepresents 1/Vmax(app) versus 1/CI from the data in Fig. 4B. Error bars
are indicated. The kinetic constants Vmax,mKAdoMet, and mKCI were calcu-
the slopes of Fig. 4B were not used to verify the kinetic con- lated from these plots.
stants obtained from Fig. 4A. However, a preliminary estimate
of their values (Slope 1/AdoMet) was used to generate a broad
range interval for the value of the inhibition constant for the De Novo and Maintenance Methylation Catalyzed by the Re-
DNA, by setting Slope 1/AdoMet = AX/VK + BC/VX + A/V(1 + combinant Human DNA (Cytosine-5) Methyltransferase—Find-
BC/AK), where A indicates KAdoMet m ; X indicates [CI]; V indi- ing a 30-fold difference in the kcat value of poly(dI-dC)·poly(dI-
cates Vmax; K indicates KiCI; B indicates KAdoMet; and C indi- dC) by the recombinant human and mouse DNMT1 prompted
cates KCIm (40). The equation is based on the assumption that us to address the question of the de novo and maintenance
the reaction is ordered, with AdoMet binding before the DNA to methylation properties of the enzyme. Two sets of oligonucleo-
the catalytic center of DNMT1. However, this assumption tides were evaluated kinetically. One set of oligonucleotides
needs to be verified. The KAdoMet is the dissociation constant for corresponds to the imprinted locus SNRPN (small nuclear ri-
AdoMet, for which the equation gives a value of ~1.0 µM. boprotein-associated peptide N) exon-1, and the other set was
KmAdoMet
, KCG , and KCI are listed in Table I. The value for from a short stretch of the FMR-1 locus (fragile X mental
m , k cat i
kcat for the recombinant human enzyme is 184 (131–237) h—1, retardation syndrome). Both DNA sequences mimic human
at least 30-fold higher than the values found from the MEL genes and are methylated during development or disease pro-
cells or recombinant mouse enzymes (11, 23). The Km value of gression. Three sets of duplex DNAs were made with either
CI was 0.5 (0.3– 0.7) µM, whereas that for the AdoMet was 7.2 both strands unmethylated or one strand methylated (hemi-
(5.3–9.1) µM. The Ki for DNA is estimated between 0.2 and 1.2 methylated). Two hemimethylated duplexes contained either
µM CI. These constants can be used as the basis for a compar- the coding (upper) or the complementary strand (lower) in
ison of different enzyme preparations. methylated form. The FMR-1 locus contained either 24 (un-
Methylation by Recombinant Human DNA Methyltransferase 33007
TABLE I
Comparison of steady-state kinetic parameters of human and murine DNMT1 using poly(dI-dC) · poly(dI-dC) as substrate
Mammalian DNMT1 kcat KAdoMet
m KmCI kcat/KmCI KCI
i Ref.
—1 6 —1 —1
h µM 10 × M h µM
Recombinant human 184 ± 75 7.2 ± 2.8 0.5 ± 0.2 377 0.2–1.2 Present work
Recombinant murine 4.5 ± 0.5 2.5 ± 0.5 1.9 ± 0.07 2.6 23
Recombinant murine (his tagged) 2.7 ± 0.5 2.4 ± 0.5 0.06 ± 0.02 45 51
Murine (MEL cells) 6.4 ± 0.3 0.4 ± 0.06 17.2 11

methylated duplex) or 12 (hemimethylated duplex) cytosine


residues available for methylation. Similarly, the SNRPN
exon-1 locus contained either 16 or 8 target cytosines (assum-
ing that the CG dinucleotide is the only target site for
methylation).
A series of double-reciprocal plots were made with six differ-
ent AdoMet or six different DNA concentrations following the
same principles described for poly(dI-dC)·poly(dI-dC). For the
unmethylated duplex, the CG concentration ranged between
0.5 and 5.0 µM, and for the hemimethylated substrate it was
between 0.1 and 1.0 µM. The AdoMet concentration was varied
from 4.0 to 15.1 µM for unmethylated DNA and 2.0 to 12.1 µM
for hemimethylated DNA. At each given DNA and AdoMet

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concentration, less than 5% of the cytosines were converted to
5 methylcytosine, and the AdoMet concentration was in vast
excess over that of the end product, AdoHcy. Double-reciprocal
plots were obtained using the SNRPN exon-1 locus either as an
unmethylated or hemimethylated substrate. With increasing
concentrations of the co-substrate, the slope of the plots de-
creased, as expected. Representative double-reciprocal plots of
SNRPN exon-1 at fixed AdoMet and at fixed DNA are shown in
Fig. 6, A and B. Replots of 1/Vmax(app) versus 1/AdoMet (from
Fig. 6B), slopes of 1/v versus 1/AdoMet (from Fig. 6B), 1/Vmax-
(app) versus 1/CG (from Fig. 6A), and slopes of 1/v versus 1/CG
(from Fig. 6A) were all linear (data not shown). kcat for the
unmethylated imprinted SNRPN exon-1 locus was about 2.3
h—1. However, the kcat value for hemimethylated SNRPN
exon-1 was 23 and 49 h—1 for the lower and upper methylated
strand duplexes, respectively (Table II). The values obtained
for KCGm were similar for both hemimethylated duplexes.
Similar experiments were carried out with the FMR-1 locus
substrate. In this case, the double-reciprocal plots and replots FIG. 6. Double-reciprocal plots for the human SNRPN exon-1.
A, fixed AdoMet plot. The AdoMet concentration was 2.0 µM (filled
were linear for (CGG·CCG)12 and (m5CGG·CCG)12 but were diamonds), 4.0 µM (open squares), 8.0 µM (filled squares), 9.8 µM (open
curved for (CGG·Cm5CG)12. Together with the data from poly(dI- circles), and 12.0 µM (filled circles). B, fixed DNA plot. The CG concen-
dC)·poly(dI-dC), these curved velocity patterns reveal that the tration was 0.15 µM (open diamonds), 0.18 µM (filled diamonds), 0.25
sequence composition and methylation status of the DNA µM (open squares), 0.37 µM (filled squares), 0.75 µM (open circles), and
template may dramatically alter the kinetics of the reac- tion. The 1.50 µM (filled circles). The fitting of the data was performed as de-
scribed (32).
kinetic constants measured for the FMR-1 locus are shown in
Table III. Unmethylated trinucleotide repeats had a very low kcat
(1.2 h—1). The kcat of hemimethylated triplet DNMT1 (32), KmAdoMet is defined as the ratio between cat k and
repeat DNA increased about 10 –20-fold, depending on the the forward rate constant for the binding of AdoMet to the
methylated strand (Table IV), as observed for the SNRPN catalytic center. For all the DNA templates tested, kcat was
exon-1 locus. Furthermore, since kcat measures the rate of greater for the hemimethylated substrates than for the un-
dissociation of the products (methylated DNA and AdoHcy) methylated ones, which increases KmAdoMet in the former mole-
from the enzyme, the increases in kcat for the hemimethylated cules. However, since KmAdoMet values were lower for hemimeth-
DNA substrates are consistent with human DNMT1 having a ylated duplexes, we believe that such low KmAdoMet values are
greater affinity for hemimethylated DNA (41) than for fully due to substantially higher rates of AdoMet binding. In the
methylated DNA. Finally, the finding that the kcat values var- accompanying paper (32), we provide evidence that hemimeth-
ied 2–3-fold between the upper and the lower hemimethylated ylated DNA activates the reaction rates by binding to an allo-
strands suggests that DNA product release is additionally in- steric site in DNMT1. We propose that the consequence of such
fluenced by the sequence composition flanking the CG sub- allosteric binding is to increase the accessibility of AdoMet to
strate site. the catalytic center.
Some of the hemimethylated templates, such as SNRPN The second-order rate constant defined by the ratio of kcat/
exon-1 and FMR-1 substrates (methylated lower strand, Tables KCG
m is also a good measure of the catalytic efficiency of an
II and III), showed a lower KmAdoMet than their unmethylated enzyme. For human DNMT1 the catalytic efficiency for hemi-
counterpart. According to the reaction scheme used to derive methylated DNA was 3–10-fold higher than the corresponding
the velocity equations for the methyl transfer reaction by unmethylated DNA (Table IV). Thus we conclude that human
33008 Methylation by Recombinant Human DNA Methyltransferase
TABLE II
Steady-state kinetic parameters of recombinant human DNMT1 using
SNRPN exon-1 as substrate
Oligonucleotide duplex kcat KAdoMet
m KCG
m kcat/KCG
m

h—1 µM 106 × M
—1
h—1
Unmethylated 2.3 ± 0.1 7.2 ± 1.7 0.5 ± 0.1 4.5 ± 1.1
Methylated upper strand 49 ± 13.2 7.1 ± 2.8 1.3 ± 0.3 37.6 ± 0.3
Methylated lower strand 23 ± 3.3 1.6 ± 0.6 1.3 ± 0.2 17.3 ± 0.1

TABLE III
Steady-state kinetic parameters of recombinant human DNMT1 using
FMR-1 locus as substrate
Oligonucleotide duplex kcat KAdoMet
m KCG
m kcat/KCG
m

h—1 µM 106 × M
—1
h—1
Unmethylated 1.2 ± 0.2 3.8 ± 1.2 0.3 ± 0.1 4 ± 1.2
Methylated upper strand 8.3 ± 1.5 2.6 ± 1.5 0.2 ± 0.06 48 ± 2.2
Methylated lower strand 22 ± 3.6 1.7 ± 0.3 0.5 ± 0.1 44 ± 2.4

TABLE IV FIG. 7. Asymmetric methylations by human DNMT1. Radioac-


Preference for hemimethylated DNA by human DNMT1 tive duplex oligonucleotides were covalently cross-linked with human
DNMT1. The presence or absence of DNMT1 and/or AdoMet in the
Relative reaction is indicated with either a + or — sign above each lane. The
Oligonucleotide duplexes Relative kcat kcat/KCG
(HM/UM) a (HM/UM)
m
target sites are indicated on the top as FG, FCG, or FWG. F represents
5-fluoro-2'-deoxycytidine, and W is either A or T. DNA-protein cross-
FMR-methylated upper strand 6.8 ± 0.7 7.1 ± 0.2

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linked bands are above 175 kDa and are indicated by an arrow. Pre-
FMR-methylated lower strand 18.3 ± 1.5 11.8 ± 2.2 stained protein molecular mass markers are indicated on the right.
SNRPN-methylated upper strand 21.0 ± 4.9 8.8 ± 2.1
SNRPN-methylated upper strand 9.9 ± 1.0 3.2 ± 0.1
a
HM indicates hemimethylated, and UM indicates unmethylated. as placenta, lung, brain, and heart (14). Recently, recombinant
expression systems using COS cells (28) and baculovirus (23)
DNMT1 is catalytically more efficient on hemimethylated than have been reported for the murine DNMT1. In COS cells the
on unmethylated DNA. expression was very low, but the baculovirus system led to a
Non-CG Methylation in Vitro by the Recombinant Human much higher level of protein expression. However, even in the
DNA (Cytosine-5) Methyltransferase—The above data clearly baculovirus system, purification involved several chromato-
demonstrate that the full-length human DNMT1 prefers hemi- graphic steps leading to a reduced yield of full-length protein.
methylated DNA as a substrate. However, there is methylation The addition of a hexa-histidine tag to the N terminus of the
in mammals at non-CG sequences (42, 43). To test whether DNMT1 and nickel column affinity purification was attempted
human DNMT1 can methylate non-CG sequences, a series of (23). However, in this purification method several other pro-
FdC-containing oligonucleotide duplexes were made with teins were present after the affinity elution, and further chro-
non-CG sites such as FXG, where X was either C, A, or T and matographic purification was required. Thus we switched to an
F was 5-fluoro-2'-deoxycytidine. The complementary strand of intein-based purification system (29).2
the FdC strand was methylated to create a hemimethylated The full-length DNMT1 gene was placed in a vector such
duplex (see “Experimental Procedures”). A stable complex is that it was fused in frame to an intein and a chitin-binding
formed between FdC-containing oligonucleotide and DNMT1 domain. The fusion precursor is immobilized on a chitin matrix
only after transfer of the methyl group from AdoMet to the where the intein can be activated to catalyze its own cleavage
target cytosine. The complex results from the inability of the and release the full-length human DNMT1 protein. Although
fluorine atom on C-5 of cytosine to serve as a leaving group and the intein-chitin domain remains bound to the affinity beads,
permit release of the enzyme (9). Following SDS-polyacryl- the target protein is eluted. We have been successful in obtain-
amide gel electrophoresis of the reaction mixture, the complex ing 1 mg of protein/1.5 × 109 cells (a liter of infected SF9 cells).
can be visualized by autoradiography. This technique has been This purification protocol yields intact protein at over 95%
used successfully to cross-link Dcm DNA m5C MTase from E. purity. Often a secondary band close to 55 kDa was observed
coli (44). From the autoradiography it is clear that human which was later established to be the viral (AcNPV)-encoded
DNMT1 forms a complex with non-CG sites (Fig. 7). The effi- chitinase (data not shown). To facilitate cleavage from the
ciency of complex formation was in the order FG > FCG > purification tag, the two C-terminal amino acids were changed
FWG, where the 5' F is the methyl acceptor in each case. Hence from lysine and aspartic acid to proline and glycine. This had
we conclude from these results that human DNMT1 also no detectable effect on activity.
methylates non-CG sequences. Poly(dI-dC)·poly(dI-dC) as a substrate has been shown to
have either comparable or greater methyl group acceptance
DISCUSSION
ability than other DNAs (38). In this work, we demonstrate a
Previously, several purification attempts were described for complex behavior of the human DNMT1 with the poly(dI-
mammalian m5C MTases using human placental tissue (37), dC)·poly(dI-dC) substrate. At higher concentrations of CI, sub-
murine erythroleukemia cells (13), and murine ascites cells strate inactivation was apparent. It was suggested that mouse
(45). However, because of the small amount of the protein and DNMT1 can form reversible, multimeric complexes at a higher
the many purification steps required to approach homogeneity, protein concentration (46), and this may be the cause of the
it has been difficult to purify these proteins. The problem is inactivation. Kinetic observations by Hitt et al. (47) suggest an
compounded by the presence of a protease-susceptible domain irreversible binding of murine DNMT1 to DNA and aggrega-
in the first 300 amino acids of the N-terminal domain and the tion at high molar excess of the enzyme. However, our kinetic
large molecular mass of the m5C MTases, about 183.5 kDa (13, measurements were carried out at 2 nM human DNMT1. In this
23). This gene is selectively expressed in a few tissue types such concentration range, protein aggregation was not detected in a
Methylation by Recombinant Human DNA Methyltransferase 33009
gel shift assay (data not shown). Thus aggregation seems un- lation. However, within the nucleus, DNMT1 binds to the rep-
likely. Several other DNA substrates that we tested using the lication fork (8) along with many other factors, such as p21 and
above conditions also do not support the aggregation hypothe- proliferating cell nuclear antigen (22). Thus, the enzyme is in a
sis. However, an alternative explanation of enzyme inactiva- very different environment during DNA replication, and the
tion at high poly(dI-dC)·poly(dI-dC) concentrations via the rate of methylation in vivo may differ from that observed in
for- mation of a ternary complex consisting of DNA-enzyme- vitro.
DNA may explain our observations. This hypothesis is Apart from methylating the canonical CGs, the human
supported by the presence of a DNA-binding peptide motif, B1, DNMT1 can also methylate unusual structures such as slipped
at the N terminus of the human DNMT1 protein (48). Motif duplexes, snapbacks, and cruciforms (50). Clark et al. (42) have
B1, as well as a deleted portion of it —DB1, interacts and binds shown that mammalian cells were capable of maintaining
strongly to DNA. DB1 can bind oligonucleotide duplexes as methylation of cytosine at CNG sites within the transfected
small as 30 bp through the lysine residues (48). Thus, it is plasmids. There are also reports of non-CG methylation in the
possible that poly(dI-dC)·poly(dI-dC) may have formed hypermethylated CG promoter region of the human L1 retro-
catalytically inactive complexes with human DNMT1 at high CI transposon (43). Methylation at such sites and its maintenance
concentrations. It should be noted that proteolysis of the N were postulated to be mediated by a different enzyme(s) (25).
terminus of murine DNMT1 leads to its catalytic activation (21), We tested the ability of the recombinant human DNMT1 to
suggesting that this domain may be involved in repression of carry out non-CG methylation by substituting the target cyto-
methyl transfer. One potentially significant observation with sine with a fluorocytosine and allowing the reaction to proceed
recombinant human DNMT1 is the high turnover number in the presence of AdoMet (44). Oligonucleotide duplexes con-
compared with previous reports for poly(dI-dC)·poly(dI-dC) taining FCG or FWG sequences formed covalent complexes
(Table I). This dif- ference could be due to the inherent nature with the human enzyme, indicative of a successful methyl
of the human DNMT1 or could reflect a greater percentage of transfer event. Thus, human DNMT1 can carry out de novo and
active protein because of our purification protocol. This could maintenance methylation at the CG sites and can also main-

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also be caused by the substrate, which has a limited search space tain methylation of some non-CG sites.
for methy- lation. Our results are consistent with the kcat values Acknowledgments—We thank Prof. S. Baylin, The Johns Hopkins
of other AdoMet-dependent methyltransferases such as the University, for the gift of the cDNA clones for human DNMT1 (pBK-
bacterial m5C MTases M.HhaI, 78 h—1 on poly(dG-dC)·poly(dG- SHMT5.0-b). We thank Drs. Ming Xu and Sanjay Kumar for
dC) (9), suggestions.
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33010 Methylation by Recombinant Human DNA Methyltransferase

Recombinant Human DNA (Cytosine-5) Methyltransferase: I. EXPRESSION,


PURIFICATION, AND COMPARISON OF DE NOVO AND MAINTENANCE
METHYLATION
Sriharsa Pradhan, Albino Bacolla, Robert D. Wells and Richard J. Roberts
J. Biol. Chem. 1999, 274:33002-33010.
doi: 10.1074/jbc.274.46.33002

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This article cites 44 references, 11 of which can be accessed free at
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