Characterizing DNA methyltransferases with an ultrasensitive luciferase-linked continuous assay

Article abstract of DOI:10.1021/ac200816m

DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-l-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-³H]AdoMet. A 5′-methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1-1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter k_cat but not K_m for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.

Full text of DOI:10.1021/ac200816m

ARTICLE
pubs.acs.org/ac
Characterizing DNA Methyltransferases With An Ultrasensitive
Luciferase-Linked Continuous Assay
Ivan Hemeon, Jemy A. Gutierrez, Meng-Chiao Ho, and Vern L. Schramm*
Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx,
New York 10461, United States
S Supporting Information
b
ABSTRACT: DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the
transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the
5
-position of cytosine residues and thereby silence transcription of regulated
genes. DNMTs are important epigenetic targets. However, isolated DNMTs
are weak catalysts and are difficult to assay. We report an ultrasensitive
luciferase-linked continuous assay that converts the S-adenosyl-L-homocys-
teine product of DNA methylation to a quantifiable luminescent signal.
Results with this assay are compared with the commonly used DNA labeling
3
0
from [methyl- H]AdoMet. A 5 -methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine.
Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly
luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic
range (0.1ꢀ1000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light
output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic
properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b
activation by DNMT3L is shown to involve two distinct kinetic states that alter kcat but not K for AdoMet. The assay is shown to be
m
robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.
3
doMet methyltransferases catalyze the transfer of a methyl
group from S-adenosyl-L-methionine (AdoMet) to a methyl
Current DNMT assays are discontinuous, using [methyl- H or
14
A
methyl- C]AdoMet followed by separation of the methylated
DNA product from unreacted AdoMet. Radioactive AdoMet
assays provide high sensitivity but require safety precautions and
involve multiple sample handling procedures. Separation of DNA
and AdoMet involves selective elution of unreacted AdoMet from
a DNA-binding medium. High background signals arise from
nonspecific binding of AdoMet or AdoMet breakdown products
to the medium. Phenol/chloroform extraction followed by DNA
precipitation reduces the contaminating proteins and lipids but
acceptor. Biological methyl acceptors include proteins, lipids,
carbohydrates, oligonucleotides, and various small molecule
metabolites. DNA (cytosine 5)-methyltransferases (DNMTs,
EC 2.1.1.37) catalyze the transfer of a methyl group from
AdoMet to a DNA substrate. The methyl group is transferred
to the 5-position of a cytosine base, usually when the cytosine
precedes a guanosine residue at so-called “CpG islands”. Methy1-
lation effectively inhibits transcription of the associated gene.
Although most DNMTs prefer to methylate CpG sites, there is
increasing evidence that they can also methylate cytosines at
4,5
6,7
introduces recovery errors. Digestion of DNA with nuclease P1
and alkaline phosphatase followed by HPLC separation of the
nucleosides allowsspecific analysisoflabeled 5-methylcytosine but
2,3
CpA, CpT, and CpC sites. Thus, DNMTs are an important
part of the mechanisms by which gene transcription is controlled
in development, differentiation, and cancer.
8
is slow and error-prone. Biotinylated oligonucleotide substrates
1
,9
facilitate DNA isolation in avidin-coated 96-well plates. How-
ever, the use of biotinylated substrates is not always desirable. A
scintillation proximity assay avoids separation of AdoMet and
DNA since DNA binds to yttrium silicate scintillant beads, but this
approach also suffers from nonspecific AdoMet binding. Fluor-
escence-based assays use an anti-AdoHcy antibody in conjunction
Human cancer cell lines possess hypermethylated CpG
islands, and it has been postulated that overmethylation de-
creases the transcription of tumor suppressor genes, promoting
1
0
2
cancer proliferation. Inhibitors of DNMTs may provide a
chemotherapeutic option by reducing CpG hypermethylation,
allowing transcription of the tumor suppressor genes and redu-
11
with AdoHcy bearing a fluorescent tag or use S-adenosyl-L-
homocysteine hydrolase (SAHH) to generate homocysteine that
3
cing tumor proliferation. Development of DNMT biology and
12,13
reacts with thiol-sensitive fluorophores.
These assays do not
inhibitor design would be facilitated by a sensitive assay that can
be used to quantitate DNMT activity and be used for inhibitor
screens. The in vitro rates of DNA methylation by DNMTs are
slow; therefore, the assay must be sensitive and also give stable
signals.
Received: April 4, 2011
Accepted: May 5, 2011
Published: May 05, 2011
r 2011 American Chemical Society
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|Anal. Chem. 2011, 83, 4996–5004

Analytical Chemistry
ARTICLE
Figure 1. Schematic overview of the AdoHcy detection assay. AdoHcy is formed from AdoMet-dependent DNA methylation via DNMT and is
converted to adenine using MTAN. The adenine is converted to AMP in an APRTase-catalyzed reaction with PRPP, then to ATP using PPDK with
phosphoenolpyruvate. ATP reacts with luciferin and O
continuously cycled to ATP.
2
in a firefly luciferase-catalyzed process to generate a stable light signal as the resulting AMP is
have a large range and require the use of thiol-free assay mixtures
reactions, the product S-adenosyl-L-homocysteine is converted
to adenine by recombinant 5 -methylthioadenosine/S-adenosyl-
12
0
or the use of anaerobic conditions. Spectrophotometric assays
have been developed for other AdoMet-dependent methyltrans-
ferases, one using SAHH and Ellman’s reagent under discontin-
L-homocysteine nucleosidase (MTAN, EC 3.2.2.9). Light pro-
duced in this assay is directly proportional to AdoHcy concen-
tration. As AdoHcy is an inhibitor of AdoMet-dependent
14
uous anaerobic conditions and another using S-adenosyl-L-
homocysteine nucleosidase to generate adenine from AdoHcy
hydrolysis which is then converted to hypoxanthine using adenine
19
methyltransferases, its removal by MTAN also prevents pro-
duct inhibition. The continuous nature of this assay eliminates
product separation and sample preparation steps. Recombinant
catalytic domains of human DNMT3a (CD-DNMT3a) and
DNMT3b (CD-DNMT3b) are compared to the bacterial M.
SssI CpG methyltransferase and to recombinant human
DNMT1. The full-length human DNMT1 is thought to be
responsible for maintaining the methylation pattern of the
1
5
deaminase. These cannot accommodate low AdoMet or DNA
concentrations due to low sensitivity and also encounter inter-
fering absorption from DNA substrates. Another method involves
liquid chromatography ꢀtandem mass spectrometry (LCꢀMS/
16
MS) and has been applied to the activity of DNMT1.
The luciferase-based luminescent assay described here for
AdoMet-dependent methyltransferases can be used under con-
tinuous or discontinuous conditions in high-throughput plate
formats. This assay evolved from our adenine detection assay
developed for ribosome-inactivating proteins, including ricin
1
,20
genome during cell division and genome replication.
DNMT3a and DNMT3b are thought to play roles in de novo
methylation of the genome during embryonic development and
1,20
have also been implicated in cancers. We have characterized
activation of human DNMT3b by DNMT3L, a catalytically
inactive accessory protein that interacts with DNMT3a and
DNMT3b to enhance their catalytic activities. Assays in the
presence of high concentrations of the pyrimidine analogues
5-azacytidine and 5-azacytosine demonstrate robust responses of
this assay for screening chemical libraries.
1
7
A-chain and subsequently adapted to protein methyl-
1
8
trasferases. The common feature of these luciferase-linked
0
assays is the conversion of adenine to adenosine 5 -monopho-
sphate (AMP) by adenine phosphoribosyl transferase
(
APRTase, EC 2.4.2.7) using 5-phospho-R-D-ribosyl-1-pyropho-
0
sphate (PRPP). AMP is converted to adenosine 5 -triphosphate
(
(
ATP) in a single step by pyruvate orthophosphate dikinase
PPDK, EC 2.7.9.1) using phosphoenolpyruvate (PEP) and
’
RESULTS AND DISCUSSION
inorganic pyrophosphate; the resulting ATP reacts with firefly
luciferase to generate light.
17,18
This assay has a wide operating
Detection and Quantification of AdoHcy. Recombinant
range and is sensitive to low adenine concentrations. For DNMT
MTAN from S. enterica was used to hydrolyze AdoHcy to give
4
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Analytical Chemistry
ARTICLE
respectively. The 286 amino acid C-terminal catalytic domains
were expressed for kinetic analysis. These domains were mod-
ified to contain an N-terminal His tag and a thrombin cleavage
6
site. The catalytic domain was identified through sequence
alignments of DNMT3a and DMNT3b from humans and mice
which gave excellent homology in the C-terminal domains. All
sequences contained a cysteine residue at the position for the
proposed catalytic nucleophile (e.g., 706 in human DNMT3a).
The C-terminal regions of human DNMT3a/3b also aligned well
with bacterial (cytosine 5)-methyltransferases. The bacterial
DNMTs are smaller than mammalian DNMTs and consist
primarily of the catalytic domain. The catalytic domains of mouse
DNMT3a and DNMT3b have been previously expressed in
21,22
E. coli.
Synthetic gene constructs for CD-DNMT3a and CD-
DNMT3b were transformed into strains of E. coli competent
cells, and it was found that BL21 star (DE3) cells afforded the
highest yield of CD-DNMT3a while BL21 (DE3) pLysS cells
were best for CD-DNMT3b expression. Expression was best at
low temperatures (18 °C, 18 h) as much of the protein was
expressed into inclusion bodies at 37 °C and could not be
refolded after purification under denaturing conditions. Purifica-
tion with a room-temperature Ni-NTA agarose column eluted
CD-DNMT3a with 500 mM imidazole and CD-DNMT3b with
Figure 2. AdoHcy assay and standard curve: (A) 36 pmol AdoHcy in
1
00 mM EDTA after washing away non-His -tagged proteins
6
2
5 μL of DNMT assay buffer and 25 μL of continuous assay buffer
with 100 mM imidazole. Pure fractions were dialyzed into a
buffer containing 0.1% DTT as significant protein precipitation
occurred during dialysis in the absence of DTT.
measured in the kinetic acquisition mode of the luminometer at room
temperature. The signal reached a maximum value within 90 s.
(
B) AdoHcy standard curve obtained by mixing 25 μL of AdoHcy
DNMT3L was also expressed since this protein has been
reported to interact with DNMT3a and DNMT3b in both full-
length and catalytic domain constructs and to enhance their
solutions (0.24ꢀ800 pmol) in DNMT assay buffer with 25 μL of
continuous assay buffer. After equilibration at room temperature for
3
to 5 min, the continuous luminescence output was measured. The
23
slope of the line is 0.909 ( 0.023, corresponding to a slope of 2.5%
error in these measurements.
catalytic activities. Expression of DNMT3L from a synthetic
gene construct in E. coli BL21 (DE3) cells gave protein expres-
sion after 3 h induction at 37 °C and 2.5-fold increased expres-
sion after overnight expression at 18 °C. DNMT3L was purified
by elution from a Ni-NTA agarose column with an imidazole
gradient.
adenine, the purine base converted to ATP in continuous assay
buffer. The product ATP is linked to luciferase to generate a light
signal and regenerate AMP (Figure 1). AdoHcy (25 μL) solu-
tions were mixed in 96-well microplates with continuous assay
buffer (25 μL) containing MTAN concentrations varying from
Human DNMT1 was also expressed. It is known to be
significantly more active than DNMT3a, DNMT3b, or their
mixtures with DNMT3L. As the catalytic domain of DNMT1 has
6
μM to 125 nM and the luminescence measured in the
24
continuous kinetic acquisition mode. In this concentration range,
been reported to be inactive, the full-length 1620 amino acid
construct was expressed in SF9 cells using a baculovirus-based
expression system. Purification of DNMT1 was also achieved by
elution from a Ni-NTA agarose column with an imidazole
gradient.
MTAN hydrolyzed AdoHcy solutions to completion in less than
2
min; thus, 125 nM MTAN was used for subsequent assays.
MTAN does not hydrolyze AdoMet, thus optimal and/or
variable AdoMet concentrations are compatible with this assay.
A response curve was constructed by mixing 25 μL of the 2ꢁ
continuous assay buffer with 25 μL of AdoHcy standard solutions
in a 96-well plate (Figure 2A). Luminescence measured in the
continuous acquisition mode gave maximum luminescence from
the AdoHcy signal within 2 min. Light output as a function of
AdoHcy (picomoles) gave a linear correlation (Figure 2B). Alter-
natively, all solutions could be mixed in several wells, and the
luminescence measured after several minutes of incubation in the
discontinuous acquisition mode of the luminometer to construct
the same curve (Figure 2B). The AdoHcy standard curve matched
that obtained from adenine standard solutions, indicating that the
coupling between AdoHcy and adenine is complete in terms of the
DNMT Activity in Continuous Assays. The use of HPLC-
purified AdoMet improved the accuracy and reproducibility of the
AdoHcy production assays. AdoMet is unstable at neutral and
alkaline pH and degrades via an intramolecular reaction giving
0
5 -methylthioadenosine (MTA) and homoserine lactone and a
2
5
hydrolytic reaction giving adenine and S-pentosylmethionine.
MTA is a substrate for MTAN and is hydrolyzed to give adenine.
Thus, both breakdown pathways produce intermediates that
contribute to the background in the luciferase-linked DNMT
assay. Commercial AdoMet sources contain up to 10% MTA
and/or adenine. HPLC purification reduced these to below 0.5%
and can be efficiently performed on large scales using standard
HPLC equipment. The purified AdoMet can be stored at ꢀ80 °C
for >6 months without loss.
1
7
ATP yield. The range of this assay is broad as solutions contain-
ing 0.1ꢀ1000 pmol of AdoHcy in volumes of 50 μL fall within the
detection range of the standard curve.
AdoMet background decomposition rate for initial rate mea-
surements is accomplished by incubating the complete reaction
mixture without DNMTs for 3 min while measuring the
Expression of Human DNMTs. Full-length human DNMT3a
and DNMT3b are composed of 912 and 853 amino acids,
4
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ARTICLE
background luminescence. This approach gives a stable linear
background signal to be subtracted from the rate following
DNMT addition (see ordinate intercepts in Figure 3A). The
DNMT reaction is initiated by the addition of the enzyme
followed by a 3 min incubation and 3 min measurement of
luminescence. The order of DNA and DNMT addition can be
reversed to allow background measurement of the AdoMet and
DNMT solutions. The 3 min incubation after reaction initiation
eliminates a lag phase prior to formation of a linear signal. The lag
phase is attributed to enzyme-DNA binding and conformational
changes since preincubating DNMTs with the DNA substrate
shortens or eliminates the lag phase. Increasing the amounts of
coupling enzymes and/or the amount of luciferase coupling
agents (ATPlite) does not affect the lag phase. The reproduci-
bility of continuous assay rates (multiple repeats of the same
assay conditions) was (10% of the mean.
Bacterial M.SssI CpG methyltransferase is a well-characterized
DNA methyltransferase and was also used to benchmark the
0
assay. The commonly used synthetic oligonucleotide poly(2 -
0
deoxyinosinic-2 -deoxycytidylic acid) (poly(dIdC)) was selected
as the DNA substrate. It is reported to have high activity with
26
DNMTs. Initial reaction rates were measured with a constant
amount of poly(dIdC) while AdoMet concentration was varied in
order to measure its K (Figure 3B). Conversion of AdoHcy to a
m
luminescent signal has the added benefit of removing the reaction
product as AdoHcy is known to inhibit DNMTs. Reaction rates
with 1 μg of poly(dIdC) and 4 U of M.SssI CpG methyltransfer-
ase (45 nM, in 50 μL reaction mixtures) were plotted against
AdoMet concentration and fit to the MichaelisꢀMenten equa-
tion (Figure 3B). This gave a K for AdoMet of 3.5 ( 0.32 μM
m
ꢀ
1
and a k of 179 ( 2.8 h , consistent with reported values
cat
6
(Figure 3B, Table 1). Thus, the error in rate measurement (k )
cat
in a practical kinetic assay of the method is (1.6%.
In our hands, CD-DNMT3a and CD-DNMT3b alone gave a
catalytic activity of <0.50 pmol/h, near the lower sensitivity of
27
this assay and similar to most literature reports. The activities of
the CD-DNMT3a and CD-DNMT3b constructs increased in the
presence of 1:1 molar ratios of DNMT3L when the complexes
were preincubated for 40 min at room temperature. Convenient
enzyme concentrations were explored by measuring reaction
rates at 10 μM AdoMet and 0.5 μg of poly(dIdC) while varying
the concentration of CD-DNMT3b þ DNMT3L in 50 μL assay
mixtures. Enzyme concentration over the range of 0.2ꢀ4.0 μM
gave initial rates proportional to the enzyme concentration.
MichaelisꢀMenten curves with varying AdoMet and poly(dIdC)
concentrations were analyzed with 500 nM CD-DNMT3 and
DNMT3L (parts A and B of Figure 4, respectively). The dIdC
concentration of poly(dIdC) was calculated based on an average
polymer length of 2100 bp (1200ꢀ3000 bp range) giving an
6
average double-stranded molecular weight of 2.57 ꢁ 10 g/mol
and a dIdC base pair concentration of 0.82 nmol dIdC/μg
poly(dIdC) (7.8 nM DNA polymer in the assay). The Michaelisꢀ
Menten equation cannot be used to analyze the kinetic constants
since enzyme (0.2ꢀ4.0 μM) greatly exceeds substrate concen-
tration (7.8 nM (poly(dIdC)). When the amount of enzyme
used is significant relative to the concentration of substrate,
corrections can be made by fitting to the Morrison equation
Figure 3. Continuous assay measurements for initial rate reactions and
for substrate saturation of M.SssI CpG methyltransferase. (A) Initial
rates of the M.SssI CpG methyltransferase reaction with increasing
AdoMet concentrations. (B) Initial reaction rates fit to the Michaelisꢀ
Menten equation for M.SssI CpG methyltransferase (45 nM) and 1 μg
of poly(dIdC) at room temperature. The standard errors of 9.1% for
28
which includes terms for substrate depletion:
pffiffiffiffiffiffiffiffiffiffiffi
2
ð½Eꢂ þ ½Sꢂ þ K Þ ꢀ ð½Eꢂþ½Sꢂ þ K Þ ꢀ 4½Eꢂ½Sꢂ
m
m
v ¼ Vmax
2½Eꢂ
K
m
and 1.6% for kcat in this traditional steady-state kinetics assay are in
The equation accounts for substrate bound to the enzyme,
allowing valid analysis of kinetic constants. These fits gave an
AdoMet K of 69 ( 40 nM, a poly(dIdC) K of 330 ( 58 nM,
agreement with published values for direct spectroscopic recording
assays.
m
m
Table 1. Kinetic Parameters for DNMTs in the Luciferase-Coupled Continuous Assay
AdoMet
m
a
DNA
m
b
ꢀ1
cat (h )
enzyme
K
(nM)
K
(nM)
Vmax (pmol/h)
k
c
M.SssI
3500 ( 320
2660 ( 750
<50
N/D
404 ( 6
726 ( 75
9.5 ( 0.6
24.6 ( 1.6
179 ( 3
DNMT1
837 ( 404
28.1 ( 2.9
0.39 ( 0.02
1.00 ( 0.07
d
CD-DNMT3aꢀDNMT3L
CD-DNMT3bꢀDNMT3L
19 ( 36
69 ( 40
329 ( 58
a
Near saturating poly(dIdC) concentration. dIdC base pair concentration = 16.4 μM. All assays were conducted at room temperature of approximately
2 °C. Near saturating AdoMet concentration used: 35 μM for DNMT1, 10 μM for CD-DNMT3a and CD-DNMT3b in complexes with DNMT3L.
Not done. Two factors contribute to the error; the lowest practical poly(dIdC) concentration (82 nM) is high relative to the K
b
2
c
d
m
value, and the high
enzyme/substrates ratio requires fitting to the Morrison equation (see text) where the propagation of errors from equation complexity give rise to the
large data fitting error.
4
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ARTICLE
Figure 5. Initial rates for 258 nM DNMT1, 6 μg of poly(dIdC), 300 μL
total volume at room temperature and at 37 °C measured by discontin-
uous assay: (A) 682 nM AdoMet, luciferase-linked AdoHcy assay;
3
(
B) 672 nM [methyl- H]-AdoMet, radiometric filter paper assay.
enzyme complexes, CD-DNMT3a-DNMT3L and CD-DNMT3b-
DNMT3L, have substantially lower K values than the bacterial
m
M.SssI CpG methyltransferase. The AdoMet Km for CD-
DNMT3a-DNMT3L is more than 70-fold lower than that for
M.SssI CpG methyltransferase. Both human CD-DNMT3-
DNMT3L mixtures have kcat values 2ꢀ3 orders of magnitude
lower than that for the M.SssI CpG methyltransferase.
Figure 4. Continuous assay measurements on human DNMTs at room
temperature. Initial rates fit to the Morrison equation for significant
depletion of substrate by enzyme: (A) 1 μg of poly(dIdC), 492 nM
CD-DNMT3bꢀDNMT3L, varied AdoMet concentration; (B) 10 μM
AdoMet, 492 nM CD-DNMT3bꢀDNMT3L, varied poly(dIdC) con-
centration; (C) 10 μM AdoMet, 490 nM CD-DNMT3aꢀDNMT3L,
varied poly(dIdC) concentration. The error bars represent the standard
deviation of 2 or 3 individual rate measurements for each rate
measurement.
Full-length human DNMT1 prefers hemimethylated DNA as
a substrate in which one of the two strands contains methylated
cytosine; however, it is more active on unmethylated poly(dIdC)
DNA than CD-DNMT3a or CD-DNMT3b in complex with
26
DNMT3L. DNMT1 (516 nM) initial rate data were fit to the
Morrison equation to give a AdoMet Km of 2.7 ( 0.7 μM, a
ꢀ
1
poly(dIdC) Km of 840 ( 400 nM, and a kcat of 28 ( 3 h
(
Table 1). These values are similar to those for the bacterial M.
SssI CpG methyltransferase, although its kcat value is 6.5 times
ꢀ
1
and kcat of 1.0 ( 0.07 h (Table 1). For CD-DNMT3a with
DNMT3L (490 nM) and 1 μg of poly(dIdC), the reaction is
saturated with AdoMet concentrations above 100 nM. Initial
rates with decreasing AdoMet concentrations indicated a Km
value of less than 50 nM. These data fit well to a two-term
Morrison equation containing two Km and two Vmax terms,
suggesting that the AdoMet concentration may also alter the
CD-DNMT3aꢀDNMT3L interaction in addition to catalytic
site saturation.
greater than that for human DNMT1, consistent with reported
6,29
values.
The results with several DMNTs demonstrate the effective-
ness of this luciferase-linked assay for characterizing DNA
methyltransferase reactions. Measurement of highly- and less-
active DNA methyltransferases is facilitated by the broad dy-
namic range of the assay. Finally, product inhibition by AdoHcy
is prevented by its continual removal.
DNMT1 Activity Using Discontinuous Assays. Discontin-
uous assays have applications in end point assays and inhibitor
screens. They are also suited to the measurement of kinetic data
under experimental conditions not suited to luminometer reac-
tion conditions.
Data obtained with 490 nM CD-DNMT3a-DNMT3L, 10 μM
AdoMet, and varying poly(dIdC) concentrations were fit to the
Morrison equation to give a poly(dIdC) K of 19 ( 36 nM and a
m
ꢀ
1
k
cat
of 0.39 ( 0.02 h (Figure 4C; Table 1). The relatively large
error in the K value results from propagation of errors from the
Initial reaction rates for human DNMT1 were measured at
room temperature and at 37 °C. Assays used 300 μL reaction
mixtures containing 682 nM AdoMet, 6 μg of poly(dIdC),
and 258 nM DNMT1. Aliquots were removed every 2 min
m
repeated terms in the Morrison equation. [When the same data is
inappropriately fitted to the MichaelisꢀMenten equation, an
apparent (but incorrect) K of 201 ( 50 nM is obtained.] The
m
5
000
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ARTICLE
(
2ꢀ10 min) and were quenched with 100 mM HCl. Aliquots
low AdoMet concentrations, and the assay is suitable for auto-
mated screening. Initial rate plots (e.g., Figure 5A) could be
constructed under various substrate concentrations using the
luciferase-linked assay under discontinuous conditions in order
to obtain kinetic data as in Figure 4.
from a no-enzyme control were treated in the same manner. The
aliquots were neutralized with 100 mM KOH, mixed with one
volume of 2ꢁ AdoHcy to ATP conversion buffer, and incubated
at room temperature for ∼3 min. Aliquots of these samples were
mixed with four volumes of ATPlite in a 96-well plate format to
convert the ATP into a luminescent signal. After incubation
at room temperature for ∼3 min, the luminescence of each
sample was measured and an initial rate plot was constructed
3
Parallel reactions were performed with 672 nM [methyl- H]-
AdoMet to compare the initial rates from the luciferase assay with
4,5
those obtained from the radiometric filter paper assay. The
luciferase-linked assays were performed at the same AdoMet
concentration. Aliquots were sampled at 2 min intervals (2ꢀ8 min)
and spotted on 2.3 cm diameter Whatman DE81 filter paper
circles. After drying (30 min), the circles were washed with
0.2 M NH HCO , water, and ethanol. The dried circles were
(
Figure 5A). Initial rates measured in relative light units (RLU)/
second were converted to units of picomole/hour with an
AdoHcy standard curve constructed under the same assay
conditions (Table 2). The rate at 37 °C was greater than that
at room temperature, 7.5 vs 6.3 pmol/h, respectively. These rates
are lower than the 30 pmol/h obtained from an identical reaction
performed using the continuous luciferase-linked assay, thus the
components of the continuous assay favor the methyltransfer
reaction. The discontinuous rates were readily measured even at
4
3
analyzed for radioactivity in a scintillation counter. Background-
correction for a no-enzyme control gave the reaction rates
(Figure 5B). Initial rates were converted to picomoles/hour
3
using [methyl- H]AdoMet standards (Table 2). Initial rates of
11.6 pmol/h at 22 °C and 13.0 pmol/h at 37 °C are similar to
those obtained in the luciferase assays.
Table 2. Initial Rates of DNMT1 Methylation of Poly(dIdC)
in Luciferase-Coupled (Using AdoMet) or Radiometric
Effects of DNMT3L on CD-DNMT3b activity. The kinetic
pattern for DNMT3L activation of DNMTs has not been
resolved. One report using a truncated version of DNMT3a
showed that maximum stimulation of DNMT activity occurred
when equimolar amounts of DNMT3a and DNMT3L were
3
(Using [Methyl- H]-AdoMet) Assays
a
assay
rate (pmol/h)
5
discontinuous luciferase 22 °C
discontinuous luciferase 37 °C
radiometric 22 °C
6.3 ( 1.2
7.5 ( 0.8
preincubated for 5 min or 2 h. Another report showed increased
stimulation with DNMT3L concentrations to 70-fold excess with
mouse CD-DNMT3a and to 20-fold excess with mouse CD-
DNMT3b by examining initial rates under a single set of
11.6 ( 0.7
13.0 ( 0.4
radiometric 37 °C
23
a
nonsaturating substrate concentrations. A third report showed
full stimulation of full-length mouse DNMT3a and DNMT3b
DNMT1 was present at 258 nM in the presence of 0.2 μg/μL
poly(dIdC). AdoMet was present at 682 nM for luciferase-linked assays
and at 672 nM for radiometric assays.
3
with a 10-fold excess of DNMT3L. Most recently, the
Figure 6. MichaelisꢀMenten curves for AdoMet binding at 1 μg of poly(dIdC) and 500 nM CD-DNMT3b with varying DNMT3L concentrations:
A) 124 nM DNMT3L, (B) 252 nM DNMT3L, (C) 492 nM DNMT3L, (D) 996 nM DNMT3L, (E) 4940 nM DNMT3L, and (F) 9800 nM DNMT3L.
Data are fit to the Morrison equation to account for significant enzyme concentrations. Maximum catalytic rates are shown in Figure 7.
(
5
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Analytical Chemistry
ARTICLE
6
the 5-azapyrimidines (2.6 ꢁ 10 -fold molar excess), neither
showed inhibition. Free 5-azapyrimidines are unlikely to be
involved in the mechanism of action of these agents. The robust
action of the luciferase assay even in the presence of millimolar
pyrimidine analogues supports the use of this assay for the
purpose of screening chemical libraries.
’
CONCLUSIONS
A light-based continuous detection assay has been developed
for the detection of AdoHcy released from DNA methyltransfer-
ase reactions. This assay uses an MTAN that hydrolyzes AdoHcy
to give adenine and S-ribosylhomocysteine but does not interact
with AdoMet. Adenine is converted to ATP using APRTase and
PPDK. ATP is quantitated by a luciferase-catalyzed reaction with
luciferin. This reaction produces a stable luminescent signal since
the AMP product is cycled to ATP to sustain the signal. This
assay has the added benefit of removing AdoHcy from reaction
mixtures to prevent product inhibition. The assay is capable of
detecting subpicomole quantities of AdoHcy and has a broad
dynamic range from 0.1 to 1000 pmol of AdoHcy. This con-
tinuous assay is a unique development for AdoMet-dependent
DNA-methyltransferases because of the sensitivity, dynamic
range, and link to light production with recycling of AMP for
stable light signal. The range of applications is broadened by use
under both continuous and discontinuous conditions, as needed
for chemical library screening and hit validation.
Figure 7. Increasing DNMT3L concentration increases activity of CD-
DNMT3b in a biphasic manner. Vmax values calculated from Michaelisꢀ
Menten curves for AdoMet binding with 1 μg of poly(dIdC). The data
was fitted to the equation for two independent saturable sites for
DNMT3L.
processivity of full-length human DNMT3a was shown to be
30
increased in a 1:1 ratio with DNMT3L. Here we provide a
titration of CD-DNMT3b with DNMT3L to demonstrate the
utility of the luciferase assay system in kinetic activation
experiments.
A 1:1 stoichiometric mixture of DNMT3L and CD-DNMT3a
or CD-DNMT3b was used in the work reported above. Kinetic
plots for AdoMet substrate saturation with near-saturating DNA
concentrations and varied ratios of DNMT3L/CD-DNMT3b
are useful to assist in the resolution of the mechanism of
activation questions.
With ratios of 0.25 to 20, DNMT3L was incubated with 500 nM
CD-DNMT3b protein and the results used to construct kinetic
plots for activation and AdoMet saturation (Figure 6). Data were
The utility of the assay is demonstrated by the ability to
observe distinct kinetic parameters for the activation of CD-
DNMT3b at low and high concentrations of DNMT3L. Exam-
ples of the assay in inhibitor screening are provided by appro-
priate catalytic responses in the presence of 5-azacytidine and
5
-azacytosine at concentrations in excess of 1 mM.
fit to the Morrison equation, giving K values that did not change
m
but Vmax values that increased with increasing DNMT3L ratios
’
MATERIALS AND METHODS
(Figure 7). This change in Vmax was biphasic, increasing in a near-
linear response at low DNMT3L concentrations and with con-
tinued activation at higher ratios but with a lower response rate.
The cocrystal structure has shown that the CD-DNMT3aꢀ
DNMT3L complex exists as in Figure 6 as a heterotetramer in
which two CD-DNMT3aꢀDNMT3L complexes interact at the
M.SssI CpG methyltransferase and AdoMet were purchased
from New England Biolabs (Ipswich, Massachusetts). AdoMet
was further purified by reverse-phase HPLC using a Waters C18
column (4.6 mm ꢁ 250 mm), 20% acetonitrile at 0.5 mL/min.
0
0
Poly(2 -deoxyinosinic-2 -deoxycytidylic acid) was purchased
from Sigma (Ashland, Massachusetts; catalog no. P4929). Firefly
luciferase ATP assay kit (ATPlite) and Betaplate Scint scintilla-
tion fluid were purchased from Perkin-Elmer, as was S-adenosyl-
2
2
CD-DNMT3a/CD-DNMT3a interface. Although this kinetic
pattern of Figure 7 is possible from a number of different protein
association models, the data fit well to the equation for two
kinetically significant complexes with Km1 = 160 ( 5 nM and
Vmax1 = 30 ( 5 pmol/h for the first complex. The second complex
was not sufficiently saturated to give quantitative kinetic constants
in this experiment, but visual inspection suggests a Km2 above
3
L-[methyl- H]methionine with a specific activity of 70.8 Ci/
mmol (Waltham, Massachusetts). EDTA-free complete protease
inhibitor cocktail tablets and PhosSTOP phosphatase inhibitor
tablets were purchased from Roche Applied Science
(Indianapolis, Indiana). Recombinant S. cerevisiae APRTase
9
μM and a Vmax2 above 100 pmol/h.
5
-Azacytidine and 5-Azacytosine Effects. The luciferase-
bearing an N-terminal His tag and recombinant Clostridium
symbiosum PPDK were prepared as previously described.
6
1
6
based DNMT assays reported here have been developed in 96-
well plates and are well-suited to larger high-throughput formats.
We explored the sensitivity of this assay to interference by
inhibitor fragments using two pyrimidine base analogues. Inhibi-
tion of DNA methylation in vivo is caused by 5-azacytidine and
-azacytosine. The mechanism is known to involve metabolism
to the deoxynucleoside triphosphates followed by incorporation
into DNA and covalent inactivation of the DNMT enzymes.
However, it is also possible that direct inhibition of DNMT
activity could occur in the presence of the inhibitors.
The 5-azapyrimidines were tested for inhibition of the CD-
DNMT3bꢀDNMT3L complex with DNMT3L in 1-fold excess
to CD-DNMT3b. Under assay conditions with up to 1.28 mM of
Synthetic DNA constructs coding for DNMTs and MTAN were
purchased from DNA 2.0 (Menlo Park, California). The Gate-
way LR Clonase II enzyme mix was purchased from Invitrogen
(Carlsbad, California). Plasmid DNA was isolated and purified
using the Qiagen HiSpeed plasmid midi kit (Valencia,
California). Ni-NTA agarose was purchased from Qiagen. Corn-
ing 96-well nonbinding surface microplates were purchased
through Fisher Scientific (Pittsburgh, Pennsylvania). Protein
concentrations were measured by Bradford assay in comparison
with bovine serum albumin (BSA) standards obtained from
Bio-Rad (Hercules, California). All other reagents used were
from Fisher Scientific or Sigma-Aldrich and were the highest
5
31
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Analytical Chemistry
ARTICLE
quality available. Luminescence was measured using a Glomax
expression under IPTG induction, and glycerol stocks were
prepared from the best expression clones. For protein expression,
LB medium was seeded with an overnight culture of BL21 (DE3)
grown in the presence of ampicillin (100 μg/mL) and incubated
at 37 °C with shaking at 250 rpm. Induction was initiated at
9
6-well luminometer from Promega (Madison, Wisconsin).
Scintillation counting was performed using a TriCarb 2910TR
liquid scintillation counter from Perkin-Elmer.
Expression and Purification of Human DNMT1. Human
DNMT1 expression constructs containing an N-terminal His6
tag and an rTEV protease cleavage site were subcloned from the
pFastBac HTa plasmid (a generous gift of Prof. Keith D.
Robertson, University of Florida). Polymerase chain reaction
of human DNMT1 cDNA was used to incorporate an SpeI
A
600 = 0.6 with 0.5 mM IPTG for 4 h. Cells were harvested by
centrifugation at 4000 rpm for 20 min and resuspended in lysis
buffer (40 mM TEA-HCl pH 7.8, 300 mM NaCl, 0.2 mg/mL
lysozyme, 0.1 mg/mL DNaseI, protease inhibitors). Cells were
lysed with three rounds of sonication for 15 s, each round with
1 min cooling time between bursts. Cell debris was removed by
centrifugation at 16 000 rpm for 30 min, and the cleared super-
natant was loaded on a 5 mL Ni-NTA column equilibrated in
lysis buffer. The column was washed with 5 column volumes of
10 mM imidazole in running buffer (20 mM TEA-HCl pH 7.8,
300 mM NaCl). Elution was done using a step gradient of 25 mM
imidazole in running buffer with 250 mM imidazole in the final
fraction. Fractions were analyzed by SDS-PAGE, and pertinent
fractions were pooled together and dialyzed overnight in 20 mM
TEA-HCl, pH 7.8, 50 mM NaCl. Glycerol was added to 10% of
the volume prior to storage at ꢀ80 °C.
0
0
restriction endonuclease site at the 3 end (sense primer, 5 -
0
CCAACTCGGTCCGAAACCATGTCGTACTACC-3
antisense primer, 5 -GGCTCACTAGTTGTACTTCTCGA-
CAAGCTTGGTACCGC-3 . The restriction sites are under-
and
0
0
lined). This PCR product was digested with RsrII and SpeI and
ligated downstream of the polyhedrin promoter of a pFastDual
vector that contains green fluorescent protein cDNA down-
stream of the p10 promoter (the generous gift of Dr. Kartik
Chandran, Department of Microbiology and Immunology,
32
Albert Einstein College of Medicine, ). The ligation of human
DNMT1 was verified by DNA sequencing and contains an I311
V mutation in human DNMT1 isoform b (GenBank Identifier
AdoHcy to ATP Conversion Buffer. The 2ꢁ AdoHcy to ATP
conversion buffer was prepared as previously described with
4
503351). A complete description of the expression and pur-
16
ification of DNMT1 is provided in the Supporting Information.
Expression and Purification of Human DNMT3a Catalytic
Domain (CD-DNMT3a). The catalytic domain of human
DNMT3a was identified by sequence alignment with bacterial
cytosine methyltransferases that consist mainly of catalytic
domains as well as through previous reports of the mouse
minor modifications. Briefly, 100 mL of buffer contained
100 mM Tris-acetate pH 7.7, 2 mM PEP, 2 mM sodium
pyrophosphate, 2 mM PRPP, 15 mM (NH ) SO , 15 mM
4
2
4
(NH ) MoO , and phosphatase inhibitors (Roche PhosSTOP,
4
2
4
10 tablets). A 4:1 cellulose/charcoal mixture was suspended in
water and packed into a disposable mini-column to a bed volume
of 1.5 mL, through which a 10 mL aliquot of the previously
prepared buffer was passed at 2 mL/min to remove traces of
adenine, AMP, and ATP. Treated buffer was filtered (0.2 μm)
and stored in 1 mL aliquots at ꢀ80 °C. Immediately prior to use,
the 2ꢁ conversion buffer was completed by additions to give
21
DNMT catalytic domains. A construct coding for a 286 amino
acid C-terminal domain of human DNMT3a (residues 627ꢀ912
of human DNMT3a, NCBI accession number Q9Y6K1) con-
taining an N-terminal His tag with a thrombin cleavage site was
6
purchased from DNA 2.0 in a pDONR221 Gateway donor vector
(Invitrogen). A complete description of the expression and
final concentrations of 10 mM MgSO , 6 U APRTase, 18 U
4
purification of CD-DNMT3a is provided in the Supporting
Information.
PPDK, and 250 nM MTAN and an increase in volume of 14 μL.
DNMT Kinetics Using Continuous Luciferase Assay. Con-
tinuous assay buffer (2ꢁ) was prepared by adding 200 μL of
luciferin/luciferase (ATPlite) to 1 mL of the AdoHcy to ATP
conversion buffer and warmed to room temperature. To 25 μL of
this mixture was added AdoMet and the DNA substrate in
DNMT assay buffer (25 mM HEPES pH 7.6, 50 mM KCl, 5%
glycerol, 0.1% DTT) in a 96-well nonbinding surface microplate.
The mixture was incubated at room temperature for 3 min
Expression and Purification of Human DNMT3b Catalytic
Domain (CD-DNMT3b). The catalytic domain for human
DNMT3b was identified as described for CD-DNMT3a. A
construct coding for a 286 amino acid C-terminal domain of
human DNMT3b (residues 568ꢀ853 of human DNMT3b,
NCBI accession number Q9UBC3) containing an N-terminal
His tag with a thrombin cleavage site was purchased from DNA
6
0
2
.0 in a pDONR221 Gateway donor vector (Invitrogen). A
to permit any contaminating adenine, AMP, ATP, or 5 -
complete description of the expression and purification of CD-
DNMT3b is provided in the Supporting Information.
methylthioadenosine (MTA) to be converted to a stable back-
ground light signal as measured in the kinetic acquisition mode of
the luminometer for 3 min. DNMT was added to give a final
volume of 50 μL. After 3 min, the luminescent signal was
measured for the following 3 min. Reactions with the less
active DNMT3LꢀCD-DNMT3a and DNMT3LꢀCD-DNMT3b
complexes were performed in triplicate. Initial rates were calcu-
lated by subtracting the background rate from that obtained after
DNMT addition. Initial rates were fit to the Morrison equation
for tight binding substrates with the exception of data obtained
while using M.SssI CpG methyltransferase which were fit to the
MichaelisꢀMenten equation. The Morrison equation was used
as high enzyme concentrations were necessary and it could no
longer be assumed that the free substrate concentration was
equal to the total substrate concentration. The kinetic parameters
Km and Vmax were obtained from these fits after converting the
luminescent rate (relative light units/second) to an enzymatic
Expression and Purification of Human DNMT3L. A con-
struct coding for the full-length human DNMT3L (NCBI
accession number NP-037501) containing an N-terminal His6
tag with a thrombin cleavage site was purchased from DNA 2.0 in
a pDONR221 Gateway donor vector (Invitrogen). A complete
description of the expression and purification of DNMT3L is
provided in the Supporting Information.
Expression and Purification of S. enterica MTAN. The gene
encoding MTAN in Salmonella enterica was synthesized and
cloned into pDONR221 vector by DNA 2.0 (Menlo Park, CA),
incorporating an N-terminal His tag sequence and terminal attB
6
R
sequences. The gene was swapped into pDEST14 vector (amp )
using Invitrogen Gateway Cloning Technology (Carlsbad, CA),
and successful clones were transformed into BL21 (DE3)
competent cells. Several colonies were screened for protein
5
003
dx.doi.org/10.1021/ac200816m |Anal. Chem. 2011, 83, 4996–5004

Analytical Chemistry
ARTICLE
rate (picomoles of AdoHcy/hour) using the AdoHcy standard
curve. The AdoHcy standard curve was constructed by mixing
Sciences and Engineering Research Council of Canada to I.H.
(Grant PDF-434344-2007).
2
5 μL of DNMT assay buffer containing varied concentrations of
AdoHcy with 25 μL of continuous assay buffer, incubating for
several minutes, and then measuring the luminescence of each
sample in the discontinuous acquisition mode of the lumin-
ometer. Values were background corrected using a mixture
containing no AdoHcy.
’
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(
’
AUTHOR INFORMATION
(
Corresponding Author
E-mail: vern@aecom.yu.edu. Phone: (718)430-2813. Fax:
718)430-8565.
7
*
(
(
(31) Champion, C.;Guianvarc’h, D.; S ꢀe namaud-Beaufort, C.; Jurkowska,
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ACKNOWLEDGMENT
(32) Li, J.; Rahmeh, A.; Morelli, M.; Whelan, S. P. J. J. Virol. 2008,
This work was supported by NIH Research Grant CA135405
82, 775–784.
and an NSERC Post-Doctoral Fellowship from the Natural
5
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dx.doi.org/10.1021/ac200816m |Anal. Chem. 2011, 83, 4996–5004