Methyltransferase-directed derivatization of 5-hydroxymethylcytosine in DNA

Article abstract of DOI:10.1002/anie.201007169

Sequence-specific derivatization: Enzymatic methylation of cytosine in DNA is part of an epigenetic regulatory network in vertebrates. In the absence of the methylation cofactor S-adenosyl-L-methionine, bacterial cytosine-5 methyltransferases can catalyze the condensation of aliphatic thiols and selenols with 5-hydroxymethylcytosine, a recently discovered nucleobase in mammalian DNA, to yield 5-chalcogenomethyl derivatives (see scheme).

Full text of DOI:10.1002/anie.201007169

Communications
DOI: 10.1002/anie.201007169
DNA Labeling
Methyltransferase-Directed Derivatization of 5-Hydroxy-
methylcytosine in DNA**
Zita Liutkevi cˇ i u¯ t e˙ , Edita Kriukien e˙ , Indr e˙ Grigaityt e˙ , Viktoras Masevi cˇ ius, and
Saulius Klima sˇ auskas*
The modification of cytosine by S-adenosylmethionine-de-
pendent DNA methyltransferases is part of an intricate
epigenetic regulation mechanism in vertebrates. DNA cyto-
sine-5 methyltransferases (C5-MTases) catalyze the transfer
of a methyl group from S-adenosyl-l-methionine (AdoMet or
from their target residues in vitro. Natural methylation by C5-
MTases proceeds through the direct transfer of the sulfonium-
bound methyl group from SAM to the C5 position of the
cytosine ring, which is activated by the nucleophilic addition
of a conserved cysteine residue in the enzyme to the 6-
[1]
[10–12]
SAM, 2) to the cytosine (1) residue in CpG dinucleotides.
Recent studies of genomic DNA from mouse embryonic stem
cells, neurons, and the brain found that a substantial fraction
of 5-methylcytosine (3) in CG sequences is converted into 5-
hydroxymethylcytosine (hmC, 5) by the action of 2-oxoglu-
position of the ring (Scheme 1a).
To better understand the mechanism of these novel
enzymatic reactions, we examined the behavior of hmC
residues in DNA duplexes (see Table 1) in the presence of C5-
2
+
[2–6]
tarate- and Fe -dependent oxygenases of the TET family.
As interactions of the 5-methyl- and 5-hydroxymethyl groups
Table 1: Structure of short hmC-containing duplex substrates.
[
a]
[
7,8]
Sequence
Short name
with cellular proteins in DNA are distinct,
hmC residues
may play an independent role in yet unknown epigenetic
pathways during embryonic development, brain functioning,
and cancer progression. However, further studies of these
intriguing phenomena are hampered by the lack of efficient
analytical techniques for mapping hmC residues in the
5’-TAATAATGhGCTAATAATAATAAT
3’-ATTATTACGhGATTATTATTATTA
GhGC/GhGC
5
3
5
3
’-TAGAGTATGATGMGCTGACCCACAACATCCG
’-ATCTCATACTACGHGACTGGGTGTTGTAGGC
’-TCGGATGTTGTGGGTCAGhGCATGATAGTGTA
’-AGCCTACAACACCCAGTCGMGTACTATCACAT
G*HGC/GMGC
G*hG*C/GMGC
[
8]
genome. Herein we show that C5-MTases can direct the
condensation of exogenous thiols and selenols with hmC in
DNA to yield the corresponding 5-chalcogenomethyl deriv-
atives. These transformations open new possibilities for the
sequence-specific derivatization and analysis of this newly
discovered epigenetic mark in mammalian DNA.
[
a] M, 5-methylcytosine; H, 5-hydroxymethylcytosine (5; synthetically
incorporated); h, 5-hydroxymethylcytosine (introduced by the enzymatic
hydroxymethylation with formaldehyde and M·HhaI). M·HhaI/M·SssI
target sites are highlighted in gray, target nucleotides are underlined,
polymerase-extended nucleotides are italicized, and P-labeled cytosine
nucleotides are shown in boldface.
33
The production of hmC and its homologues in vitro was
recently demonstrated by the methyltransferase-directed
addition of exogenous aliphatic aldehydes, such as formalde-
hyde (4), to cytosine; it was also found that DNA C5-
MTases are capable of removing the 5-hydroxymethyl group
MTases under a variety of reaction conditions. Remarkably,
we found that the presence of an exogenous thiol compound,
such as 2-mercaptoethanol (6), cysteamine (7), or l-cysteine
[
9]
(8), at a millimolar concentration led to the appearance of a
[
*] Z. Liutkevi cˇ i u¯ t e˙ , Dr. E. Kriukien e˙ , I. Grigaityt e˙ , Dr. V. Masevi cˇ ius,
Prof. S. Klima sˇ auskas
Department of Biological DNA Modification
Institute of Biotechnology, Vilnius University
V. A. Grai cˇ i u¯ no 8, 02241 Vilnius (Lithuania)
Fax: (+370)5-260-2116
new product at the expense of the original hmC (Figure 1).
ESI-MS and UV spectral analyses of the new compounds
showed that the thiol replaces a hydroxy group in the hmC
residue, and the sulfur atom is directly attached to the
nucleoside (see Table S1 and Figures S1 and S2 in the
Supporting Information). Taking into account the observation
that substrates containing C or mC at the target position were
completely inert in the reaction (data not shown), and on the
basis of the documented reactivity of the 5-hydroxymethyl
E-mail: klimasau@ibt.lt
Homepage: http://www.ibt.lt/dmtl_en.html
Dr. V. Masevi cˇ ius
Faculty of Chemistry, Vilnius University
Naugarduko 24, 03225 Vilnius (Lithuania)
[7,13,14]
group towards a variety of nucleophiles,
we presumed
[
**] We thank G. Urbanavi cˇ i u¯ t e˙ and G. Vainorius for enzyme prepara-
tions, and M. Krenevi cˇ ien e˙ for technical assistance. We also thank S.
Tumkevi cˇ ius, G. Lukinavi cˇ ius, D. Daujotyt e˙ , and A. Petronis for
fruitful discussions. This research was supported by the Lithuanian
State Science and Study Foundation (P-07003), the Research
Council of Lithuania (student research fellowship to I.G.), the FP7-
REGPOT-2009-1 program (project 245721 MoBiLi), and the US
National Institutes of Health (1R21HG005758).
that addition of the thiol occurs at the 5-hydroxymethyl group
to yield 5-(2-hydroxyethyl)thiomethylcytosine (13), 5-(2-ami-
noethyl)thiomethylcytosine (14), and S-(5-methylcytosinyl)-
l-cysteine (15) residues in DNA, respectively (Scheme 1).
The identity of the isolated nucleoside 13 was confirmed by
direct chromatographic comparison of its nitrous acid deam-
ination product with a chemically synthesized 5-(2-hydroxy-
ethyl)thiomethyl-2’-deoxyuridine (see Figure S3 in the Sup-
porting Information). Similar reactivity but even at lower
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201007169.
2
090
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Angew. Chem. Int. Ed. 2011, 50, 2090 –2093

sulfide,
sodium
azide,
potassium bromide, or
sodium iodide, showed no
detectable reaction.
Besides
another
M·HhaI,
bacterial C5-
MTase, M·SssI, was exam-
ined and also showed
clearly detectable catalytic
activity at its target sites
(see Figure S4 in the Sup-
porting Information). The
generality of this phenom-
enon indicates that one role
of the enzyme is to direct
the reaction by flipping out
and exposing in the cata-
lytic center a residue that
occurs at its target posi-
[11]
tion.
Moreover,
we
found that the reaction
requires the presence of
the catalytic cysteine resi-
due in the enzyme (Fig-
ure 1a), a result that sug-
gests a covalent reaction
intermediate (Scheme 1).
The most straightfor-
ward mechanism is a direct
SN2 attack of the nucleo-
phile at the protonated 5-
hydroxymethyl
group.
However, this mechanism
does not agree with our
finding that the rate of
adduct formation is fairly
independent of whether a
thiol or the corresponding
selenol was used in the
reaction. On the other
Scheme 1. Transformations of a target residue by DNA cytosine-5 methyltransferases. a) Biological methylation
hand,
a seleno product
by C5-MTases occurs through an S 2 reaction between an activated cytosine intermediate (ACI) and the
N
always prevailed over
a
cofactor SAM (2) to give a covalent 5,6-dihydrocytosine intermediate (CDI) and then 5-methylcytosine (mC, 3).
The activated cytosine residue can also participate in reversible addition reactions (red-colored route) with
formaldehyde (4) to yield 5-hydroxymethylcytosine (5, hmC). The latter compound, including hmC residues in
natural DNA, can undergo further methyltransferase-directed condensation (green-colored route) with thiol or
selenol reagents (6–12) to give stable 5-alkyl chalcogenomethyl derivatives (13–19). b) Proposed mechanism
of nucleophile condensation at the target hmC (5) residue catalyzed by a C5-MTase. The reaction is thought to
proceed via a covalent enamine intermediate (CEI) followed by either a highly active 5-methylene intermediate
thio product even in the
presence of an excess
amount of thiol (see Fig-
ures S5 and S6 in the Sup-
porting
Information).
These observations clearly
point to a mechanism in
which the formation of an
active intermediate is the
slow rate-limiting step,
(
AMI) or a bicyclic sulfonium intermediate (CSI), which would then undergo the fast addition of a nucleophilic
reagent, such as 7. The C5-MTase and its catalytic moieties are shown in blue; boxed areas denote species
and reactions within the catalytic center of the enzyme.
concentrations was observed (Figure 1) with selenols, such as
selenocysteamine (11) and l-selenocysteine (12). 5’-Deoxy-5’-
thioadenosine (10), which can be regarded as a simple
cofactor mimic owing to a potential anchoring interaction of
the adenosine moiety with the enzyme, was also reactive at
submillimolar concentrations. Other types of nucleophiles,
such as hydrazine, p-nitrophenol, phenol, methanol, sodium
whereas the relative strength of the nucleophile determines
the nature of the final product. On the basis of previously
[
15]
proposed mechanisms for thymidylate synthase and deoxy-
[
16]
cytidylate hydroxymethylase,
one could assume that the
reaction proceeds by an acid-assisted dehydration at the 5-
hydroxymethyl group to give a highly electrophilic 5-meth-
ylene intermediate (activated methylene intermediate, AMI;
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Communications
Figure 1. Methyltransferase-directed coupling of thiols and selenols with 5-hydroxymethylcytosine residues in DNA. a) TLC analysis of a modified
3
3
P-labeled hmC nucleotide. DNA containing hmC at the target site of M·HhaI was treated with 12 mm l-cysteine, 12 mm l-selenocysteine, 12 mm
cysteamine, 12 mm selenocysteamine, 150 mm 2-mercaptoethanol, or 0.5 mm 5’-thio-5’-deoxyadenosine in the presence of 200 nm M·HhaI (wild
type (WT), a catalytic C81S mutant, or both) for 1.5 h at room temperature. Modified DNAs were digested to 5’-mononucleotides, analyzed by
TLC, and autoradiographed. b) Reversed-phase HPLC analysis of modified 2’-deoxynucleosides. A GhGC/GhGC duplex (13 mm) was treated with
an exogenous reagent as above (50 mm l-cysteine, 50 mm l-selenocysteine, 12 mm cysteamine, 12 mm selenocysteamine, 0.5 mm 5’-thio-5’-
deoxyadenosine, 500 mm 2-mercaptoethanol, or 50 mm dithiothreitol (DTT)) in the presence of 13 mm M·HhaI for 1 h at room temperature.
Modified DNAs were digested to nucleosides and analyzed by HPLC–MS methods. Small arrows indicate peaks corresponding to modified
nucleosides.
Figure 2. Sequence-specific covalent derivatization and labeling of hmC-containing DNA. a) General approach to the selective derivatization and
labeling of hmC-containing DNA with a reporter group (shown as a ball). DNA bases in the HhaI target site are shown as gray sticks.
b) Fluorescence labeling of a pUC19 plasmid containing hmC residues at the HhaI target sites. The DNA was amino-derivatized by treatment with
l-cysteine in the presence of M·HhaI, and the derivatized DNA was labeled with fluorescein N-hydroxysuccinimidyl ester, fragmented with
R·FspBI, and analyzed by agarose gel electrophoresis. Fluorescein imaging of the labeled fragments was performed with a 473 nm laser scanner
(
right panel); bulk DNA fragments were visualized after staining with ethidium bromide (left panel). In each case, lane 1 is the control with
M·HhaI omitted. c) A mixture of a hmC-containing and non-hmC DNA fragments was treated with selenocysteamine or cysteamine in the
presence of M·HhaI (no M·HhaI in the control), followed by labeling with biotin N-hydroxysuccinimidyl ester. Streptavidin-coated magnetic beads
were added and then washed to remove unbound DNA. Fragment recovery was determined by on-bead real-time qPCR analysis.
[
17]
Scheme 1b). However, this high-energy exomethylene com-
pound could in principle be avoided by an intramolecular
attack of the enzyme-borne C6-bound sulfur atom onto the
protonated 5-hydroxymethyl group. This reaction would give
a bicyclic sulfonium intermediate (CSI) containing a four-
membered thietane ring. This route requires a substantial
degree of conformational plasticity in the active site for the
catalytic sulfur atom to approach the exocyclic target. Such
conformational flexibility can be predicted from molecular-
dynamics studies of M·HhaI,
but whether the proposed
bicyclic structure is well-compatible with the active sites of
[
11]
other C5-MTases
and related enzymes (e.g. thymidylate
synthase, deoxycytidylate hydroxymethylase) remains to be
determined. In both cases, subsequent addition/attack of a
nucleophile would readily give the observed product, whereas
the addition of water would promote the reverse reaction to
give the original hydroxymethylated cytosine residue.
2
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Angew. Chem. Int. Ed. 2011, 50, 2090 –2093

The described novel reactions of C5-MTases to catalyze
the condensation of non-cofactor-like compounds with the
exocyclic hydroxymethyl group of a modified cytosine residue
provide another spectacular example of the catalytic versa-
Keywords: DNA labeling · enzyme mechanisms · epigenetics ·
methyltransferases · nucleophilic addition
.
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Received: November 15, 2010
Published online: January 26, 2011
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