(Received for publication, January 24, 1995; and in revised form, May 25, 1995)
From the
Besides modulating specific DNA-protein interactions, methylated
cytosine, frequently referred to as the fifth base of the genome, also
influences DNA structure, recombination, transposition, repair,
transcription, imprinting, and mutagenesis. DNA
(cytosine-5-)-methyltransferase catalyzes cytosine methylation in
eukaryotes. We have cloned and expressed this enzyme in Escherichia
coli, purified it to apparent homogeneity, characterized its
properties, and we have shown that it hemimethylates DNA. The cDNA for
murine maintenance methyltransferase was reconstructed and cloned for
direct expression in native form. Immunoblotting revealed a unique
protein (M
The chemistry of cytosine-5 methylation consists of transfer of
a methyl moiety from S-adenosyl-L-methionine (AdoMet) ( The
recognition sequence for DNA MTase is highly specific with almost all
cytosine methylation occurring in the duplex palindrome 5`-C-p-G-3`
(CpG). Over half of CpG dinucleotide palindromes are methylated in the
mammalian genome(26) . After semiconservative replication of
DNA, both daughter duplexes are hemimethylated, and DNA MTase, which is
localized to replication foci(27) , fully methylates the duplex
CpG dinucleotides. This process, termed maintenance methylation,
restores the parental genomic methylation pattern and is consistent
with the in vitro propensity of the DNA MTase for
hemimethylated sequences(28, 29) . DNA MTase can
also methylate certain CpGs that are not in a hemimethylated
configuration, a process referred to as de novo methylation.
Although the mechanisms for de novo methylation are not
completely understood, a number of studies have reported the appearance
of newly methylated CpG dinucleotides in the
genome(29, 30, 31, 32) . Only one
gene encoding mammalian DNA MTase has been found, and maintenance
methylation and de novo methylation are generally believed to
be catalyzed by a single enzyme(33, 34) . Several
studies have noted the appearance of de novo methylated
cytosines in genomic regions containing preexisting methylated
cytosines (i.e. methylation spreading) such as occurs in newly
integrated viral DNA in the
genome(31, 35, 36, 37) . Since
cytosine methylation can affect the DNA binding of certain
transcriptional regulatory factors, the introduction of additional
methylated cytosines within gene regulatory sequences may influence
gene expression(35) . This spreading of cytosine methylation in
gene regulatory sequences has been implicated in the gene silencing
characteristic of fragile X syndrome(38, 39) ,
cellular senescence (22) , and X chromosome
inactivation(13) . The importance of cytosine methylation in
general and the DNA MTase in particular has led us to express this
enzyme in Escherichia coli and to further study its
mechanisms. Although the cloned cDNA for murine DNA MTase (33) has been expressed in mammalian COS cells (40) ,
we report the first successful expression and purification of
catalytically active mammalian DNA MTase in E. coli, providing
a potential means for preserving native methylation patterns of cloned
DNA in this widely used and simplified system. The purification to
apparent homogeneity of DNA maintenance methyltransferase overexpressed
in E. coli will facilitate mutational analysis of this enzyme
and may allow its large scale production for crystallography. Studies
of the effects of the recombinant methyltransferase on the prokaryotic
genome and cellular processes will be useful in further elucidating the
biological significance of DNA methylation.
Figure 1:
Schematic illustration of expression
vector pTOT1 containing the cDNA for maintenance DNA MTase. Overlapping
coding sequences for the DNA MTase (pMG and pR2K, kindly provided by
Timothy Bestor, Columbia University) were endonuclease digested,
ligated, amplified, and cloned into the pKK223-3 prokaryotic
expression vector downstream of the tac promoter and ribosome
binding site (see ``Experimental Procedures''). Depicted
sequences are the 5` and 3` junctions of the DNA MTase insert within
the cloning vector. The T7 promoter, M13 primer sequence, and
termination region (ribosomal terminator) are also
illustrated.
Figure 5:
Partial purification of DNA MTase using
hemimethylated oligodeoxynucleotide substrate. Approximately 10 liters
of E. coli cells transformed with pTOT1 were induced and lysed
as indicated under ``Experimental Procedures.'' DNA MTase was
partially purified by pooling chromatography fractions on the basis of
methylating activity (i.e. transfer of radioactive methyl
moieties to trihemimethylated oligodeoxynucleotide substrate (see Table 1for structure)). A, DEAE-Sephacel (closedcircles, 2 µg of trihemimethylated oligonucleotide as
substrate; opencircles, 2 µg of
poly(dI
The S1
lysate was dialyzed 3 h with two changes of 6 liters of dialysis/column
buffer (20 mM Tris-HCl, pH 7.8, 5 mM dithiothreitol,
10% glycerol, 5 mM EDTA, and protease inhibitors as for the
lysis buffer). The dialyzed S1 solution was diluted with an equal
volume of column buffer and loaded onto a DEAE-Sephacel column (2.5
For ammonium sulfate precipitation,
pooled active fractions from DEAE chromatography were diluted with 1
volume of column buffer and brought to 30% ammonium sulfate with gentle
stirring over 10 min followed by continued stirring over 20 min on ice.
The mixture was centrifuged for 20 min at 10,000
We chose to express the maintenance DNA MTase in its native
form to allow its use in future in vivo studies (e.g. preserving methylation patterns of cloned DNA) without potential
interference with activity or DNA binding from a fusion product.
Plasmid pTOT1 was constructed to express the native DNA MTase from the
strong inducible tac promoter (Fig. 1). Immunoblotting
kinetic studies for DNA MTase indicated full expression of this enzyme
within 3 h of IPTG induction (data not shown). We cloned pTOT1 into mcr
Figure 2:
Expression of mammalian DNA MTase in E. coli. Lysates from E. coli cells transformed with
either pKK223-3 (control vector lacking the DNA MTase insert) or
pTOT1 (expression vector containing the DNA MTase insert) were resolved
by 5% SDS-PAGE, transferred to nitrocellulose, and probed with DNA
MTase polyclonal antibody. LaneM, prestained high
molecular weight protein marker; lane1, control
lysate from cells transformed with pKK223-3; lane2, expression lysate from cells transformed with pTOT1.
The unlabeledarrow indicates the novel protein (M
E. coli cells expressing mammalian DNA
MTase do not appear to grow as well as cells containing the control
vector and typically require 3.5 h to reach an A
Figure 6:
Methyl transfer by the recombinant DNA
MTase. A, ethidium bromide-stained agarose gel. A 100-µl
assay mix containing 1.66 µM
[methyl-
To assess enzymatic catalysis by the cloned DNA
MTase, the DNA methylating activity (as measured by transfer of
tritiated methyl groups from AdoMet to DNA) of DEAE-purified fractions
was compared for cells transformed with pTOT1 and pKK223-3 (Fig. 3). The DEAE columns were simultaneously chromatographed
and eluted with a salt gradient. Peak methylating activity for the
fractions from the pTOT1-transformed cell lysates eluted in the range
of 100-150 mM NaCl, consistent with results of the DNA
MTase purified from mammalian
cells(29, 30, 48) . No obvious methylating
peak was seen for the DEAE-chromatographed lysates of
pKK223-3-transformed control cells. Immunoblots performed on the
pTOT1 DEAE fractions indicated a M
Figure 3:
Correlation of DNA MTase activity with
methyltransferase immunoblotting. Individual 5-liter cultures of mcr
The cloned DNA
MTase was purified to apparent homogeneity by assaying for methylating
activity in a three-column system based on protein charge (DEAE), size
and shape (Sephadex), and DNA-affinity (DNA cellulose) (Fig. 4).
Due to the presence of various inhibitory substances in crude fractions
and lability of the enzyme (49) , meaningful estimates of total
purification factor could not be obtained consistent with reports by
others(29, 30, 50) . Gel filtration yielded a
single peak of methylating activity in the M
Figure 4:
Purification of DNA MTase to apparent
homogeneity. Expression of the DNA MTase construct (pTOT1) was induced
in 12 liters of E. coli culture, pelleted, and lysed, and the
DNA MTase was purified on the basis of assaying for methylating
activity. A, DEAE-Sephacel fractionation (closedcircles, assayed with nonmethylated duplex 60 mer (see Table 1for DNA structure) as substrate; opencircles, control assay lacking DNA template; opentriangles, 0-400 mM salt gradient). Active
fractions were pooled and ammonium sulfate-precipitated for gel
filtration (see ``Experimental Procedures''). B,
Sephadex G-150 chromatography depicting methylating activity (closedcircles) and calibration with gel filtration
protein standards (closedsquares). C, DNA
cellulose chromatography of active fractions pooled from gel filtration (closedcircles, methylating activity of
nonmethylated duplex 60-mer; opentriangles,
0-400 mM salt gradient). Assays were performed as
described under ``Experimental Procedures'' with 5 µg of
DNA where indicated. D, silver stain of pooled active
fractions from DNA cellulose (5 µg of protein loaded). Arrow indicates purified M
Partially purified recombinant DNA MTase was used for comparison of
substrate preference with increasing DNA MTase purity, assessment of
relative effectiveness of purification steps, estimates of solubility
and degree of expression of the cloned DNA MTase in E. coli,
and DNA substrate analysis studies (Fig. 5). A hemimethylated
oligodeoxynucleotide was synthesized containing methyl moieties at
approximately 15-base pair intervals for use as substrate in DNA MTase
assays (see ``Experimental Procedures'' for chemical
synthesis and Table 1for structure of hemimethylated
oligodeoxynucleotide). Preferential transfer of radioactive methyl
moieties to the oligodeoxynucleotide substrate containing
hemimethylated CpG sites over the control lacking substrate was
apparent after DEAE purification (Fig. 5A), and this
ratio improved with gel filtration (Fig. 5B). Ethidium
bromide staining of agarose gels indicated minor amounts of large
molecular weight E. coli genomic DNA present after DEAE
purification (data not shown), accounting for the slight activity of
control assays lacking oligonucleotide substrate (Fig. 5A). The chemically synthesized hemimethylated
oligodeoxynucleotide underwent greater methylating activity in DEAE and
gel filtration fractions than the highly methylatable de novo substrate, poly(dI The
pooled active fractions as well as the crude lysates were assessed on
polyacrylamide gels for protein content and purity (Fig. 5C). The soluble (S1) and insoluble (S2) SDS-PAGE
crude lysate fractions were estimated for percent DNA MTase by scanning
stained gels (see ``Experimental Procedures''). The DNA MTase
comprised approximately 2% (range of 1.0-3.0%) of total E.
coli protein in the S1 fraction and about 0.3% (range of
0-0.53%) for the insoluble S2 fraction, indicating that
approximately 85% of the enzyme is expressed in soluble form (see Fig. 5C for comparison of S1 and S2 fractions). The
overall expression of DNA MTase in these cells is about 2.5% of total E. coli protein. By contrast, mammalian cells contain a mean
of 0.05% DNA MTase of total human placental protein (see
``Experimental Procedures'' under ``Gel Scanning of
SDS-PAGE'' and (46) ). To demonstrate that the
recombinant DNA MTase is indeed active with a preference for
hemimethylated DNA, we reacted the partially purified enzyme with
oligodeoxynucleotides in the presence of radioactive AdoMet, resolved
the samples on agarose gels, and subjected the gels to autoradiography (Fig. 6). The gel-isolated hemimethylated oligonucleotide
produced the most intense band on autoradiography, demonstrating
preferential transfer of methyl moieties to hemimethylated CpGs. Some
radioactivity was apparent in the otherwise identical nonmethylated
oligodeoxynucleotide (i.e.de novo methylation), and
this activity was greater than that for the identical fully methylated
oligonucleotide containing no methylatable CpGs. Thus it is apparent
that the recombinant DNA MTase transfers methyl moieties directly to
these oligodeoxynucleotides with a preference for hemimethylated CpG
sites and with a much lower propensity for nonmethylated CpG sites.
Very little methylation appears to occur at sites other than CpG (Fig. 6B, lane4). To further
characterize the enzymatic activity of the DNA MTase purified from E. coli, we quantitated in assays the methyl receptivity of
otherwise identical oligonucleotides differing only in placement of
methyl moieties (Table 1). These analyses utilized the more
purified gel filtration fraction (Fig. 5C) containing
no evidence of contaminating E. coli DNA. Table 1shows
that the hemimethylated oligodeoxynucleotide substrate received the
most radioactive methyl transfer catalyzed by the recombinant DNA MTase
consistent with the DNA MTase partially purified from mammalian cells (51) . Also similar to the mammalian cell enzyme, nonmethylated
oligonucleotides can undergo de novo methylation, and
sequences containing no methylatable CpGs (i.e. premethylated
at all CpG sites) are poor templates for the DNA MTase (Table 1),
demonstrating its strong preference for cytosine methylation
specifically in CpG
dinucleotides(29, 30, 51, 52) . A
duplex trimethylated oligodeoxynucleotide containing only two de
novo methylatable CpGs on each stand (Table 1) is more
receptive to de novo methylation (22.9 pmol/h/CG) than an
otherwise identical nonmethylated oligonucleotide containing five de novo methylatable CpGs on each stand (7.3 pmol/h/CG),
indicating enhanced de novo methylation of a premethylated
oligodeoxynucleotide containing methylatable CpGs. The widely-used techniques of DNA cloning and PCR
amplification strip mammalian genomic DNA of its original cytosine
methylation. DNA that lacks its native cytosine methylation pattern may
give different results in mobility shift analysis, endonuclease
digestions, and other procedures analyzing its properties and behavior.
We developed the idea that the methylation pattern of cloned DNA could
be preserved in host bacteria expressing the maintenance DNA MTase.
However, the cDNA for this enzyme has previously been expressed only in
mammalian cells (COS-1)(40) . Whereas this may be of use in
studying the effects of variations in DNA MTase levels in mammalian
cells, we chose to clone and express DNA MTase in E. coli. We
developed this system not only for its possible use in maintaining
methylation patterns of cloned DNA in bacteria but also because of the
widespread use of E. coli as a protein expression system, the
simplification of cell culture and purification processes, the
potential of large scale production of the enzyme for crystallography,
and the facilitation of mutagenesis studies of this enzyme. The
known potential for de novo methylation and methylation
spreading by the DNA MTase (29, 30, 35) could
be a factor in preserving methylation patterns of genomic DNA in this
system; however, both of these processes occur in proportion to greater
DNA MTase levels (48, 51) and number of cell
generations(31, 35) . Modulating the DNA MTase
expression by limiting IPTG induction and minimizing cell culturing
times may be useful approaches for reducing the possibility of de
novo methylation. Analysis of the cloned product with
methylation-sensitive isoschizomers (44) or methylation
sequencing (53, 54) would be prudent to assess the
possibility of ectopic methylation. Previously it was thought that
the mammalian DNA MTase might be toxic to E. coli since de
novo methylation of the E. coli genome may activate the mcr system leading to DNA degradation(47) , even
though the mammalian DNA MTase is primarily a maintenance
methyltransferase and appears to de novo methylate only as a
secondary function(29, 30) . In order to circumvent
this potential problem, we cloned the reconstructed murine MTase cDNA
in mcr Studies of the effects of expression of the
cloned mammalian DNA MTase on the E. coli genome, on the
control of cellular processes in E. coli, and on replication
rates as well as cell viability may contribute to understanding the
control mechanisms of this enzyme and its biological significance. A
number of prokaryotic cellular control processes could be affected by
expression of this recombinant enzyme in these cells such as the
transcription of key regulatory genes, DNA repair, replication, and
recombination. We have previously reported several theoretical
molecular mechanisms of cellular senescence(22) , a hallmark of
which is reduced replicative capacity, and have suggested that de
novo methylation by the DNA MTase may contribute to this
phenomenon in aging eukaryotic cells (22) . Prokaryotic cells
do not senesce(22) , and studies are in progress analyzing the E. coli cells now expressing this protein for evidence
suggestive of senescing cells (e.g. morphological changes,
slowing of cell replication) and the DNA MTase as one of the putative
``mortality gene'' products. The maintenance DNA MTase,
purified from mammalian cells, is highly susceptible to proteolytic
degradation (34) and loss of enzyme activity due to its
lability(29, 30) . Moreover, the DNA MTase is present
in very limited quantities in mammalian cells(46) . The
expression of the DNA MTase in E. coli and purification of
this enzyme to apparent homogeneity may help overcome some of these
problems. It is generally known that the use of E. coli allows
rapid, easy growth of large numbers of cells with less endogenous
protein heterogeneity and bypassing of nuclear isolation protocols. In
the case of this specific enzyme, its purification from E. coli may also be facilitated by its relatively large size compared with
most E. coli proteins, allowing more effective size separation
and reducing the risk of proteolytic degradation and loss of enzyme
activity. Whatever the choice of purification protocol, the expression
of mammalian DNA MTase in E. coli should allow greater
availability of purified enzyme. All of the properties of the purified
recombinant DNA MTase examined in this study including relative
molecular mass, elution in salt gradients, affinity for DNA,
immunoreactivity, and substrate preference are consistent with the
known properties of the enzyme purified from mammalian cells
(29-30, 33-34, 49, 51-52). Although it is
generally thought that the eukaryotic DNA MTase is capable of
maintenance and de novo methylation without assistance from
associated mammalian proteins or factors, this important question is
still not fully resolved(55) . Purification of the mammalian
enzyme has helped address this issue, but minor contaminants that
assist the DNA MTase could still be present in apparently pure
fractions. Our studies indicate that the enzyme is indeed capable of
both types of DNA methylation. The expressed product in E. coli was originally derived from a single mammalian gene(33) ,
and when this cDNA is expressed in E. coli and purified, it
can perform both maintenance and de novo methylation of DNA.
Whatever other proteins may be involved in the eukaryotic methylation
process, it is clear that the essential features of maintenance and de novo methylation are not dependent upon associated proteins
unique to the mammalian replication apparatus. Similar to the
mammalian DNA MTase isolated from mammalian cells (56, 57, 58) , the recombinant enzyme
purified from E. coli has a preference for hemimethylated CpG
dinucleotides, has a tendency to de novo methylate DNA, and
transfers methyl moieties at very low levels in substrates not
containing methylatable CpG dinucleotides. Although some cytosine
methylation can occur in other dinucleotides in the mammalian genome
containing cytosine in the 5`
position(31, 59, 60) , and such activity has
occasionally been reported to be at relatively high
levels(59) , our studies with the recombinant enzyme indicate
that this occurs only very rarely in oligodeoxynucleotides containing
these dinucleotides. The mechanisms for the propensity of the enzyme
to methylate in regions already containing methyl moieties (i.e. genomic methylation spreading) are not fully
understood(35) . These studies indicate enhanced de novo methylation of oligodeoxynucleotides containing preexisting methyl
moieties, which suggests in vitro methylation spreading. A
more detailed study of methylation spreading in vitro will be
reported elsewhere. Currently, work is aimed toward preserving
methylation patterns of cloned DNA using our expression system. Other
intended studies are the effect of expression of the mammalian MTase on
control of biological processes in prokaryotic cells, further
delineation of the functional domains of the maintenance
methyltransferase in mutagenesis studies, and large scale production of
this enzyme for crystallography. Finally, studies are in progress
focusing on a more extensive analysis of the molecular mechanisms of
methylation spreading using the defined in vitro oligodeoxynucleotide system reported in this initial study.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 190,000) not present in
control cells. The mostly soluble overexpressed protein was purified by
DEAE, Sephadex, and DNA cellulose chromatography. Peak methylating
activity correlated with methyltransferase immunoblots. The purified
enzyme preferentially transferred radioactive methyl moieties to
hemimethylated DNA in assays and on autoradiograms. All of the examined
properties of the purified recombinant DNA methyltransferase are
consistent with the enzyme purified from mammalian cells. Further
characterization revealed enhanced in vitro methylation of
premethylated oligodeoxynucleotides. The cloning of
hemimethyltransferase in E. coli should allow facilitated
structure-function mutational analysis of this enzyme, studies of its
biological effects in prokaryotes, and potential large scale
methyltransferase production for crystallography, and it may have broad
applications in maintaining the native methylated state of cloned DNA.
)to carbon 5 of the pyrimidine ring of cytosine. This
simple one-carbon transfer, catalyzed by DNA
(cytosine-5-)-methyltransferase (DNA MTase), is ubiquitous, affecting
approximately 5
10
cytosines/mammalian diploid
nucleus (1) . Cytosine methylation is the most common
modification of DNA found in nature and has been implicated in the
control of developmental processes(2) , DNA
repair(3, 4, 5) , chromatin
organization(6, 7, 8) ,
transcription(9, 10, 11) , X chromosome
inactivation(12, 13) ,
transposition(14, 15) , recombination(16) ,
mutagenesis(17, 18) , replication(19) , and
genomic imprinting(20) . DNA MTase has been shown in mice to be
essential for embryonic survival (21) and has been proposed to
play a role in general biological processes such as cellular
aging(22) , carcinogenesis(23) , human genetic
diseases(24) , and evolution(17, 25) .
Bacterial Strains and Plasmids
For standard
transformations E. coli Sure cells (mcrA, mcrCB
, mrr
, hsd
,
recB
,
lacI
; Stratagene) were used
routinely. The prokaryotic expression vector pKK223-3 was
obtained from Pharmacia Biotech Inc. The cDNA for murine DNA MTase
(EMBL accession 14805 (corrected version)) was kindly provided by
Timothy Bestor (Columbia University) as overlapping coding sequences
(pMG and pR2K) cloned into pBluescript SK
M13
(33) . pMG includes all of the sequence
from the EcoRI linker at the 5` terminus of the cDNA clone to
a BglII site near the 3` end of coding(33) . pR2K
contains the sequence between the unique XhoI site at
nucleotide 3138 and an Eco47III site just downstream of the
AATAAA polyadenylation signal.
Plasmid Construction
The identity of pMG and pR2K
was verified with endonuclease digestion. Each plasmid contained an
internal XhoI site in the DNA MTase coding sequence as well as
a 3` XhoI site in the pBluescript sequence(33) . Both
plasmids were digested with XhoI and gel-purified, and the XhoI-XhoI sequence of pR2K was ligated into the
digested and gel-purified pMG plasmid lacking this segment. This fused
the coding sequences at the XhoI site (nucleotide 3138)
without alteration of the original sequence as confirmed with extensive
restriction digests. The newly formed plasmid containing the entire DNA
MTase coding sequence in pBluescript was used as template for PCR
amplification (20 cycles) of the coding sequence and 3`-untranslated
region using Vent DNA polymerase (New England Biolabs) and Perfect
Match DNA polymerase enhancer (Stratagene) under standard conditions
(see ``Polymerase Chain Reaction''). The proofreading Vent
DNA polymerase was used to assure amplification fidelity of the
sequence and the Perfect Match enhancer was used to facilitate PCR of
the approximately 5-kilobase pair segment. PCR amplification was chosen
as a cloning strategy since the ATG codon should be within 15 base
pairs of the unique EcoRI site in pKK223-3 for effective
subsequent ribosome binding. Since there were no unique sites in the
ATG region to allow cloning within this restricted distance from the
ribosome binding site, primers were synthesized that created a SmaI site just 5` of the DNA MTase ATG start codon and a HindIII site just downstream of the 3` terminus (sense primer,
5`-CCTTACCCGGGATGGCAGACTCAAATAGATC-3`; antisense primer,
5`-CGGTTAAAGCTTTTGTAAAACGACGGCCAGT-3`). The PCR product was digested
with SmaI and HindIII, phenol extracted, gel
purified, and ligated into pKK223-3 between its unique SmaI and HindIII sites just 3` of the ribosome
binding sequence. Restriction digests with SmaI and HindIII as well as several endonucleases at DNA MTase internal
unique sites confirmed successful cloning of the cDNA into the
expression vector within appropriate distance of the ribosome binding
site (pTOT1, see Fig. 1). Primers unique to the DNA MTase coding
sequence and internal to the original set of primers amplified the
expected fragment from the pTOT1 construct but not from the control
pKK223-3 vector (sense primer, 5`-ATGGCAGACTCAAATAGATCCCC-3`;
antisense primer, 5`-CTGGTGTGACGTCGAAGACT-3`). The constructed pTOT1
expression plasmid contains a tac promoter (hybrid of the
strong trp and lac promoters(41) ), ribosome
binding site, complete coding region of DNA MTase (4565 base pairs) and
3`-untranslated region, termination signal (rrnB), T7
promoter, M13 primer sequence, ampicillin resistance, and a pBR322
origin of replication (see Fig. 1).
Polymerase Chain Reaction
Amplifications were
conducted in a Perkin Elmer Cetus GeneAmp PCR System 9600 thermocycler.
Standard procedures were used, and cycling consisted of 1 min at 94
°C, 3 min at 55 °C, and 5 min at 72 °C. Aerosol-resistant
pipette tips were used for assembling all PCR reactions.DNA MTase Assays
Mammalian DNA MTase was routinely
assayed in a 100-µl volume containing a standard assay mix (10%
glycerol, 50 mM Tris acetate, pH 7.8, 10 mM EDTA, 2
mM dithiothreitol, 5 µg/µl RNase A, 0.7 µg/ml
pepstatin, 0.5 mM Pefabloc SC, 0.5 µg/ml leupeptin, 2
µg/ml aprotinin (protease inhibitors were from Boehringer
Mannheim)), 3 µCi of [methyl-H]AdoMet (60
Ci/mmol, ICN) at 1.5 µM final concentration, 5 µg of
DNA unless otherwise specified, and enzyme
sample(27, 29, 30, 42) . The enzyme
does not require magnesium, and 10 mM EDTA is used to prevent
any possible nuclease digestion of DNA substrates. Glycerol at 10% is
used due to the inherent lability of the DNA MTase. Incubations were at
37 °C for 1 h unless otherwise indicated. After completion of the
assay, reactions were terminated by the addition of SDS to 0.6%
followed by a 30-min incubation at 60 °C with 400 µg/ml
proteinase K(42) . Two volumes of 0.5 N NaOH were
added, and the samples were incubated at 60 °C for 10 min to
hydrolyze any remaining traces of RNA(42) . The samples were
cooled on ice, and carrier salmon sperm DNA was added (20
µg/assay). DNA was precipitated in 10% trichloroacetic acid, 5
mM sodium pyrophosphate for 15 min at 4 °C and washed 5
times on a Whatman GF/C filter with 5% trichloroacetic acid, 5 mM sodium pyrophosphate, and twice with 100%
ethanol(29, 30) . Washed filters were transferred to
5-ml Scintiverse BD (Fisher) in glass vials and counted on a
scintillation counter. One unit of DNA MTase activity is defined as the
amount of enzyme required to transfer 1 pmol of tritiated methyl groups
to DNA in 1 h(29, 30) .
DNA Substrates
Polymer
poly(dIdC)
poly(dI
dC) (Pharmacia) was dissolved in 10
mM Tris, pH 7.5, and 100 mM NaCl, heated to 45 °C
for 5 min, aliquoted at 1 µg/µl and stored at -20 °C.
Oligodeoxynucleotides were synthesized on an Applied Biosystems 380A
DNA synthesizer using standard procedures(25) . Methylated
5-cytosine (Glen Research) was added as the phosphoramidite where
indicated (see Table 1). All synthesized oligonucleotides were
gel-analyzed and used only if complete synthesis was evident. For
annealing oligonucleotides, complementary strands were mixed (500
ng/µl each) and incubated for 10 min at 75 °C in 20 mM Tris-HCl, pH 7.5, and 50 mM NaCl, slowly cooled to room
temperature, and analyzed for annealing efficiency on 3% agarose gels
stained with ethidium bromide.
SDS-Polyacrylamide Gel Electrophoresis
(PAGE)
Polyacrylamide gels (5%) were prepared and run at 50 mA.
Electrophoresis was terminated when tracking dye reached the bottom of
the gel. Where indicated, gels were stained for protein either with
silver nitrate (43) or Coomassie Brilliant Blue (44) using standard procedures.Immunoblotting
Transfer onto nitrocellulose (0.45
µm; Schleicher and Schuell) from SDS-PAGE gels was performed at 65
mA for 2 h at 4 °C on a TE Series transfer electrophoresis unit
(Hoefer Scientific Instruments) in SDS transfer buffer (44) to
facilitate transfer of high molecular weight protein. Staining for DNA
MTase was performed with the ProtoBlot Western blot AP system as
recommended (Promega) using 1:10,000 rabbit DNA MTase polyclonal
antibody (anti-pATH52, (34) ) (kindly provided by Timothy
Bestor, Columbia University), 1:7,500 antirabbit IgG AP conjugate, and
nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate.Protein Analysis
Except for column fractions
assayed for protein by absorbance at 280 nm, protein concentration was
determined using the Bio-Rad Coomassie assay kit. Standard curves were
established using -globulin.
Cell Cultures
Cultures of E. coli Sure
cells (transformed either with pTOT1 or control vector pKK223-3)
of 5 ml and 50 ml in 2YT medium supplemented with ampicillin (100
µg/ml) were successively grown to saturation from a single colony
at 37 °C. The tac promoter is not fully suppressed by the lac suppressor in this system, and some expression of the DNA
MTase occurs in the absence of
isopropyl-1-thio--D-galactopyranoside (IPTG). Large scale
cultures were inoculated with the saturated cell suspension (3.3
ml/liter) and grown at 37 °C until the absorbance at 600 nm was
approximately 0.5. At this point, the cells were induced with 1 mM IPTG. After 3 h, the cells were harvested by centrifugation (4,000
g for 15 min, 4 °C), washed once in
phosphate-buffered saline, and recentrifuged. Cells were either
immediately lysed or stored frozen at -70 °C as a cell
pellet. We found no difference in DNA MTase activity between cells
immediately lysed and those stored as cell pellets at -70 °C
overnight.
Purification
Unless noted, all procedures were
carried out at 4 °C. The washed cell pellet was resuspended in
lysis buffer (50 mM Tris-HCl, pH 7.5, 5% (v/v) glycerol, 2
mM EDTA, 1 mM 2-mercaptoethanol, 0.23 M NaCl, 0.1 mM dithiothreitol, 130 µg/ml lysozyme, 0.5
µg/ml leupeptin, 2 µg/ml aprotinin, 0.5 mM Pefabloc
SC, and 0.7 µg/ml pepstatin) at 3 ml/g of cells(45) . Cells
were blended (Waring) at low speed for 3 min, and after 20 min sodium
deoxycholate was added with stirring to 0.05%. The mixture was blended
for 30 s at low speed and sonicated (5 pulses on ice for 15 s). For
more complete DNA sheering, the mixture was blended for 30 s at high
speed. The sample was diluted with lysis buffer (4 ml/g of cell pellet)
lacking lysozyme, blended at high speed for 30 s, and centrifuged at
20,000 g for 30 min(45) . The supernatant (S1)
containing soluble protein was removed, and the pellet was resuspended
vigorously in 100 ml of high salt (0.4 M NaCl) lysis buffer
lacking lysozyme. The resuspended high salt mixture was centrifuged as
for the S1 solution. The supernatant (S2) containing protein insoluble
in 0.23 M NaCl was stored frozen at -70 °C. Since
several preliminary purifications indicated that over 80% of the DNA
MTase was present in the soluble S1 fraction and combining S1 and S2
reduced the resolution and yield of DNA MTase from E. coli,
all subsequent purifications were carried out using only the S1 lysate
(see Fig. 5C for solubility of DNA MTase).
dC)
poly(dI
dC) as substrate; opensquares, no added DNA substrate in the methylation assay; opentriangles, 0-400 mM salt
gradient). B, Sephadex G-150 chromatography of ammonium
sulfate-precipitated pooled active fractions from the DEAE column
(symbols as for DEAE chromatography (closedsquares,
column calibration using indicated gel filtration protein standards)). C, Coomassie stain of 5% SDS-PAGE gel. LaneM, molecular weight marker; laneS1, soluble crude lysate; laneS2, insoluble
crude lysate; laneD, pooled active fractions from
DEAE chromatography; laneSD, pooled active fractions
from Sephadex G-150 column (approximately 500 µg of protein loaded
in each lane). Arrow indicates the M
190,000 protein (DNA MTase).
12 cm bed volume). The eluted column was washed with 2 bed
volumes of column buffer to remove unbound protein and further eluted
with a 200-ml 0-400 mM NaCl gradient. Fractions were
collected and assayed for methylating activity as indicated above.
Active pooled fractions were stored at -70 °C in 50%
glycerol, 5 mM EDTA.
g,
and the supernatant was brought to 60% ammonium sulfate, stirred, and
recentrifuged. The 60% ammonium sulfate pellet containing the DNA MTase (30) was resuspended in 2 ml of column buffer, loaded onto a
Sephadex G-150 column (2
70 cm bed volume) and eluted. Active
fractions were pooled and stored as above. DNA cellulose (4 mg of
double-stranded DNA/g of solid, Sigma) chromatography was performed in
a 1
3-cm bed volume and eluted with a 0-400 mM NaCl gradient. Active fractions were pooled and stored at
-70 °C in 50% glycerol, 5 mM EDTA.
Gel Scanning of SDS-PAGE
To estimate percentage of
total cellular protein represented by the recombinant DNA MTase,
Coomassie-stained 5% SDS-PAGE gels were scanned on an Apple OneScanner,
plotted, and integrated for density using the Image 1.49 program on a
Macintosh IIfx computer. A total of four different protein
concentrations of the S1 and S2 lysates (representing a 4-fold
difference in total protein loaded) from two independent DNA MTase
purifications were resolved on SDS-PAGE gels, scanned, and plotted in
duplicate. The M 190,000 protein band (identified
with molecular mass markers) was integrated for density in each lane
and compared with the total integrated density of all proteins in the
same lane to obtain percentage DNA MTase of total E. coli protein. For similar determinations in the mammalian system, a
photomicrograph (kindly provided in reprint form by Steven Smith, City
of Hope National Medical Center, Duarte, CA) of a SDS-PAGE gel
resolving the crude lysate fraction of total human placental protein
containing the identified DNA MTase (46) was also gel-scanned,
plotted, and integrated for density as for the E. coli crude
lysates.
(modified cytosine restriction) cells to
prevent potential DNA degradation by the mcr system (47) . The lysed E. coli cells containing pTOT1
(expression vector) revealed a unique protein (M
= 190,000) on immunoblots probed with the DNA MTase
polyclonal antibody (Fig. 2). This protein was not present in
lysates of cells containing pKK223-3 (control vector lacking the
DNA MTase insert). The calculated molecular mass of the DNA MTase is
172,238 based on its coding sequence. However, this enzyme has been
shown previously to resolve at an apparent relative molecular mass of
190,000 on SDS-PAGE gels, which is thought to be due to
posttranslational modifications of the enzyme and/or its molecular
shape(33) .
= 190,000) not present in control cells.
Lysate samples were prepared from 1 ml of a 5-ml saturated cell
suspension (transformed either with pTOT1 or pKK223-3 and induced
with IPTG (see ``Experimental Procedures'')),
microcentrifuged 1 min at room temperature, resuspended in 100 µl
of 1
SDS gel loading buffer, heated to 100 °C for 3 min,
and loaded (10 µl) onto
SDS-PAGE(44) .
of 0.5 at 37 °C, whereas control cells reach this stage of
growth within 3 h. The pTOT1 cells produce slightly smaller colonies on
culture plates and less turbid overnight cultures compared with cells
containing the control vector (data not shown). These differences in
comparison to control cells became more pronounced as the cells were
transferred to successive culture plates over a period of several
months. To prevent progressive cellular proliferative retardation, we
periodically transformed fresh mcr
cells
with the pTOT1 expression vector. We have not yet fully quantified the
degree of apparent cellular proliferative and growth impairment. It
seems possible that its cause may be related to effects of DNA MTase
expression on the E. coli genome, although the large size of
the novel protein product itself may also be a factor. Evidence for
methylation of high molecular weight E. coli genomic DNA in vitro can be seen (see Fig. 6B), suggesting
that a similar process may occur in vivo affecting the growth
of these cells.
H]AdoMet (85 Ci/mmol) and 200 µg of
partially purified recombinant DNA MTase (fraction D, Fig. 5C) was incubated for 3 h at 37 °C with each
separate reaction either containing 5 µg of DNA or lacking added
DNA (control). Phenol-extracted reaction mixes were resolved on a 4.5%
agarose gel and stained with ethidium bromide. LaneM, molecular weight marker; lane1,
control lacking DNA; lane2, nonmethylated duplex; lane3, trihemimethylated duplex; lane4, pentamethylated (i.e. fully methylated at CpG
sites) duplex (see Table 1for DNA structures). Each lane
consisted of 1 µl of a 10-µl solution (500 ng of DNA where
indicated). B, autoradiogram of
H-labeled methyl
transfer to DNA. A different freshly prepared 4.5% agarose gel was
loaded with 7 µl of the same phenol-extracted samples depicted in A above (3.5 µg of DNA where indicated) and impregnated
with En
Hance for 3 h, soaked in 5% acetic acid for 1 h, and
dried on filter paper. The film for autoradiography was preflashed and
exposed to the gel for 14 days at -70 °C before developing. Lane1, control lacking added DNA; lane2, nonmethylated duplex as substrate; lane3, trihemimethylated duplex; lane4,
pentamethylated (fully methylated) duplex (see Table 1for DNA
structures). Minor diffusion of oligodeoxynucleotide bands due to
processing in En
Hance and acetic acid is apparent. High
molecular weight DNA in wells is considered to be in vitro methylated E. coli genomic DNA (see also gelA). Each oligodeoxynucleotide is identical except for its
methylated state.
190,000 protein
correlating with peak methylating activity (fractions 32-52;
pTOT1), which was not apparent below 100 mM salt (fractions
8-30; pTOT1) or above 150 mM salt (fraction 71; pTOT1). Fig. 3also shows that the most intense M
190,000 bands (fractions 38-42; pTOT1) correlated with
fractions having the highest methylating activity. The control DEAE
column showed no evidence of the M
190,000 protein
as indicated by the absence of this band at peak methylating activity
for the pTOT1 column (fraction 38; pKK223-3).
cells were transformed with pTOT1
(expression vector) or pKK223-3 (control vector) constructs and
induced with IPTG. Expression and control cell extracts were
chromatographed on two DEAE-Sephacel columns run simultaneously
side-by-side. Eluted fractions from each column using 0-400
mM NaCl were assayed for DNA MTase activity with 5 µg of
poly(dI
dC)
poly(dI
dC) as substrate (control assays
lacked added DNA substrate). Methylating activity (closedcircles, pTOT1-transformed cells; opencircles, pKK223-3-transformed cells) is expressed
as pmol/h of [
H]CH
incorporated by
fractions from each of the columns minus pmol/h of the assay control
(lacking added DNA) for each fraction from the respective column. (The
mean activity value for controls lacking DNA substrate in assays was
1.33 pmol/h (range = 0.44-2.83) for pTOT1 fractions and
0.70 pmol/h (range = 0.44-1.28) for pKK223-3
fractions. The mean activity value for the pKK223-3 (vector only
control) fractions containing added DNA substrate in assays was 0.56
pmol/h (range = 0.22-1.33).) The salt gradient (mM NaCl, opensquares) and protein concentration (A
, closedsquares) are also
shown (same for both columns). The inset depicts
immunoblotting, using the polyclonal DNA MTase antibody(34) ,
of DEAE-Sephacel eluted fractions (numbered at top) from the
pTOT1-transformed cells (+) or the pKK223-3-transformed
cells(-).
180,000-205,000 range, consistent with polyacrylamide gel
estimates. While size separation is efficient in this expression system
due to the relatively large size of the mammalian DNA MTase compared
with most E. coli proteins (Fig. 5C), some
protein impurities remain in the Sephadex fraction, and a final
purification based on the affinity of this enzyme for DNA is quite
effective in producing a homogeneous purification as assessed by silver
staining (Fig. 4D). Although improvements of the
purification procedure are expected to increase the yield of
recombinant DNA MTase, we recovered almost a full milligram (887
µg) of apparently pure enzyme from about 10 liters of E. coli cells. The apparently homogeneous protein exhibiting peak
methylating activity following DNA cellulose chromatography reacted
with the DNA MTase antibody on immunoblots (Fig. 4E).
190,000 protein. E, immunoblot of pooled DNA cellulose active fractions (5
µg of protein loaded). Arrow indicates purified protein
reacting to polyclonal DNA MTase
antibodies.
dC)
poly(dI
dC), indicating
preferential hemimethylation by the recombinant DNA MTase.
cells. The mcr
cells expressing DNA MTase are slightly less proliferative than
control mcr
cells (i.e. containing
the cloning vector alone), perhaps relating to de novo methylation of the E. coli genome. Transformation of the
vector into fresh mcr
cells appears to
improve cellular proliferation to near control levels. In spite of this
minor growth impairment, these cells are able to overexpress the DNA
MTase to relatively high levels compared with the levels of this enzyme
in mammalian cells.
-D-galactopyranoside.
We thank Timothy Bestor, Lucy Andrews, Steven Smith,
Gerd Pfeifer, and David Klapper for helpful discussions and Lucy
Andrews, Mike Conrad, Bradford Coffee, David Klapper, and Devon Byrd
for critical comments on the manuscript. We also thank Susan Elmore for
oligodeoxynucleotide synthesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.