RETRACTED ARTICLE: Divergent synthesis of 5-substituted pyrimidine 2′-deoxynucleosides and their incorporation into oligodeoxynucleotides for the survey of uracil DNA glycosylases

Article abstract of DOI:10.1039/d0sc04161k

Recent studies have indicated that 5-methylcytosine (5mC) residues in DNA can be oxidized and potentially deaminated to the corresponding thymine analogs. Some of these oxidative DNA damages have been implicated as new epigenetic markers that could have profound influences on chromatin function as well as disease pathology. In response to oxidative damage, the cells have a complex network of repair systems that recognize, remove and rebuild the lesions. However, how the modified nucleobases are detected and repaired remains elusive, largely due to the limited availability of synthetic oligodeoxynucleotides (ODNs) containing these novel DNA modifications. A concise and divergent synthetic strategy to 5mC derivatives has been developed. These derivatives were further elaborated to the corresponding phosphoramidites to enable the site-specific incorporation of modified nucleobases into ODNs using standard solid-phase DNA synthesis. The synthetic methodology, along with the panel of ODNs, is of great value to investigate the biological functions of epigenetically important nucleobases, and to elucidate the diversity in chemical lesion repair.

Full text of DOI:10.1039/d0sc04161k

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Divergent synthesis of 5-substituted pyrimidine 2 -
deoxynucleosides and their incorporation into
oligodeoxynucleotides for the survey of uracil DNA
glycosylases†
Cite this: DOI: 10.1039/d0sc04161k
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Ai Tran, Song Zheng, Dawanna S. White, Alyson M. Curry and Yana Cen *bc
a
a
b
b
Recent studies have indicated that 5-methylcytosine (5mC) residues in DNA can be oxidized and potentially
deaminated to the corresponding thymine analogs. Some of these oxidative DNA damages have been
implicated as new epigenetic markers that could have profound influences on chromatin function as
well as disease pathology. In response to oxidative damage, the cells have a complex network of repair
systems that recognize, remove and rebuild the lesions. However, how the modified nucleobases are
detected and repaired remains elusive, largely due to the limited availability of synthetic
oligodeoxynucleotides (ODNs) containing these novel DNA modifications. A concise and divergent
synthetic strategy to 5mC derivatives has been developed. These derivatives were further elaborated to
the corresponding phosphoramidites to enable the site-specific incorporation of modified nucleobases
into ODNs using standard solid-phase DNA synthesis. The synthetic methodology, along with the panel
of ODNs, is of great value to investigate the biological functions of epigenetically important nucleobases,
and to elucidate the diversity in chemical lesion repair.
Received 29th July 2020
Accepted 7th October 2020
DOI: 10.1039/d0sc04161k
rsc.li/chemical-science
The signicance of 5mC in transcription silencing, genomic
imprinting and cellular differentiation has been well estab-
Introduction
15,16
5
-Methylcytosine (5mC), also known as the h base, is both an lished.
However, the same cannot be said for its chemical
endogenous DNA damage product and an intermediate in relatives. Much has been debated about whether these 5mC
1
a recently identied epigenetic reprogramming pathway.
derivatives are of physiological relevance or just transient
17
Accumulating evidence suggests that active demethylation intermediates along the above-mentioned pathways. For
could involve iterative oxidation of 5mC to 5-hydrox- example, great progress has been made to map and sequence 5-
ymethylcytosine (5hmC), and then to 5-formylcytosine (5foC) hydroxymethyluracil (5hmU) and 5-formyluracil (5foU) in the
2
3,4
18,19
and 5-carboxylcytosine (5caC), mediated by ten-eleven trans- human genome.
However, there is still ongoing discussion
location (TET) enzymes, with the conversion of 5caC to C by about if these modications are just aberrant DNA damage, or
5
20–22
a putative decarboxylase (Scheme 1). Direct deformylation of they are key epigenetic marks regulating gene transcription.
5
6
3,4,23,24
foC can also lead to the restoration of cytosine (C). An alter- This is partly due to the low abundance of 5mC derivatives
native proposal involves 5mC deamination by activation- and their quick removal by DNA glycosylases, which make the
7,8
induced deaminase (AID), followed by thymine DNA glyco- interrogation of their biological functions extremely chal-
sylase (TDG)-initiated base excision repair (BER). Recent nd- lenging. Synthetic oligodeoxynucleotides (ODNs) containing
9
ings also implicate the removal of modied C or uracil (U) 5mC derivatives serve as powerful surrogates to examine the
derivatives by DNA glycosylase/BER/nucleotide excision repair chemical and enzymatic conversions of modied nucleobases,
10–14
(NER) as another potential demethylation mechanism.
and to probe the mechanism of action and substrate specicity
of DNA glycosylases. However, there are limited synthetic
25–29
methods available for accessing these ODNs.
And many of
these synthetic strategies involve the use of toxic chem-
a
Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health
Sciences, Colchester, VT 05446, USA
25–27,29
icals
and oen suffer from low yield and difficulty in
b
purication. More importantly, there is no unied method to
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond,
generate all the 5mC related phosphoramidites. Herein, we
Institute for Structural Biology, Drug Discovery and Development, Virginia report an efficient and concise strategy to synthesize 5mC,
VA 23219, USA. E-mail: ceny2@vcu.edu; Tel: +1-804-828-7405
c
Commonwealth University, Richmond, VA 23219, USA
5hmC, 5foC, 5caC, thymine (T), 5hmU, 5foU and 5-carboxylur-
acil (5caU) phosphoramidites through one common
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0sc04161k
1
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Scheme 1 Potential pathways for the oxidation, deamination and demethylation of 5mC and derivatives. 5mC, 5-methylcytosine; 5hmC, 5-
hydroxymethylcytosine; 5foC, 5-formylcytosine; 5caC, 5-carboxylcytosine; 5hmU, 5-hydroxymethyluracil; 5foU, 5-formyluracil; 5-caU, 5-
carboxyluracil.
0
intermediate, 5-iodo-2 -deoxyuridine. Subsequently, these Surveyor HPLC system with a photodiode array and a single
modied nucleobases, along with C and U, were incorporated quad mass spectrometer detector. Oligonucleotides were
into synthetic ODNs in a site-specic fashion via standard solid- synthesized with a Millipore Expedite nucleic acids synthesis
phase DNA synthesis. Ultimately, the ODNs were subjected to system (Billerica, MA). Mass spectra were obtained with
a panel of DNA glycosylases as well as mammalian cell extracts. a Bruker Autoex II MALDI-TOF mass spectrometer operated in
These enzymes and cell extracts demonstrate diverse substrate positive ion reectron mode. High-resolution mass spectra were
preference and cleavage efficiency. This survey provides the rst obtained with either a Waters Micromass Q-TOF Ultima (Mil-
comprehensive inventory of base selection for various glyco- ford, MA) or a Thermo Scientic Q-Exactive Hybrid Quadrupole
sylases. The novel divergent synthesis and chemical probes Orbitrap (Waltham, MA).
developed in the current study should facilitate the probing of
epigenetic reprogramming pathways, the biological signi-
cance of modied nucleobases and the diversity in chemical
lesion repair.
ODN synthesis and characterization
ODNs were prepared by solid-phase DNA synthesis as described
30–32
previously.
Following synthesis and deprotection, ODNs
were puried by HPLC using Hamilton PRP-1 column and
a gradient of 0 to 25% CH CN in 10 mM of potassium phos-
Experimental
Reagents and instruments
3
phate buffer (pH ꢁ 6.8). ODNs were then detritylated with 80%
aqueous acetic acid at room temperature for 30 min. Following
detritylation, they were puried by HPLC using Vydac C18
All reagents were purchased from Sigma-Aldrich (St. Louis, MO)
or Fisher Scientic (Pittsburgh, PA) and were of the highest
purity commercially available. Commercially available phos-
phoramidites and sublimed 1H-tetrazole in anhydrous aceto-
nitrile were purchased from Glen Research (Sterling, VA). Thin
layer chromatography (TLC) was performed on precoated silica
gel 60 F254, 5 cm ꢀ 20 cm, 250 mM thick plates purchased from
3
column and a gradient of 0 to 70% CH CN in water. ODN purity
33
was examined by MALDI-TOF, and the free base composition
34
was veried by HPLC, following enzymatic digestion using
3
a Aquasil C18 column and a gradient of 0 to 60% CH CN in
2
0 mM of ammonium acetate (pH ꢁ 5.5). The presence of
modied bases was also veried by GC/MS following acid
hydrolysis and conversion to the t-butyldimethylsilyl ethers.
3
2
EMD (Gibbstown, NJ). [g- P]-ATP was purchased from MP
Biomedicals (Irvine, CA). E coli UDG and human SMUG1 and T4
polynucleotide kinase were purchased from New England
Biolab (Ipswich, MA). Human TDG was purchased from Anta-
35
Expression and purication of human MBD4
gene (Santa Clara, CA). HeLa cell nuclear extract was purchased pET28-MBD4 was a gi from Cheryl Arrowsmith (Addgene
from Promega (Madison, WI). plasmid # 25155). Recombinant, N-terminally His-tagged
H, C, and P NMR spectra were obtained using a Bruker protein was expressed in E coli BL21 (DE3) Codon plus RIL
Ultrashield 300 MHz spectrometer (Manning Park, MA). HPLC cells (Agilent). The cells were culture in LB media supplemented
1
13
31
ꢂ1
ꢃ
analysis was performed with a ThermoFinnigan (Waltham, MA) with 50 mg mL kanamycin at 37 C until OD600 reached 0.6.
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ꢃ
Protein expression was induced with 100 mM of IPTG. The to a total volume of 10 mL. The reactions were incubated at 37 C
ꢃ
culture was grown for another 16 h at 15 C before the cells were before being quenched with the addition of 5 mL of 100 mM
pelleted and lysed with 4 rounds of sonication. The protein was NaOH, an equal volume of Maxam–Gilbert loading buffer and
puried using Ni-NTA resin (Thermo Fisher) affinity column 50 pmol of an unlabeled ODN as a reannealing competitor. The
and eluted with increasing concentrations of imidazole. The backbone was cleaved at the abasic site with NaOH by heating at
ꢃ
puried protein was dialyzed against a solution of 50 mM Tris– 95 C for 30 min. For ODNs containing 5foC or 5foU, the reac-
ꢃ
HCl (pH 7.5), 1 mM EDTA, 15% glycerol and 2 mM DTT. The tions were stopped by heating to 75 C for 5 min and then
ꢃ
protein was aliquote, ash frozen and stored at ꢂ80 C. Protein cooled to room temperature. Reaction mixtures were then
concentration was determined using Bradford assay. The resolved on a 18% denaturing polyacrylamide gel. The bands
protein was >95% pure as determined with SDS-PAGE gel corresponding to the substrate and product were visualized and
electrophoresis.
quantied using a Typhoon 9400 phosphorimager as described
above.
ODN labeling and annealing
0
32
5
-End radiolabeling was performed using [g- P]-ATP and T4
polynucleotide kinase under the conditions recommended by Results and discussion
the enzyme supplier. Labeled mixture was subsequently
Synthesis of phosphoramidites containing modied C or U
centrifuged through G-50 Sephadex columns (Roche) to remove
excess unincorporated nucleotide. Labeled single-stranded
ODNs were annealed to a 2-fold molar excess of unlabeled
complementary strand in 20 mM Tris–HCl (pH 8.0), 1 mM EDTA
Careful literature investigation suggests that commercially
0
available 5-iodo-2 -deoxyuridine (5IU) serves as an ideal starting
0
point for constructing 5-substituted 2 -deoxyuridine derivatives.
ꢃ
TBDMS-protected 5IU, compound 1, can be further function-
alized at the 5-position through a metal–halogen exchange
and 50 mM NaCl. The mixture was heated at 95 C for 5 min and
slowly cooled down to room temperature.
36
reaction (Fig. 1). When BuLi was used alone, deiodinated
product predominated, suggesting a competition between
deprotonation and lithium–iodine exchange. Instead, NaH was
introduced rst to remove the imide proton. Subsequently, BuLi
was added to effect the metal–halogen exchange and substitu-
tion. In previous studies, the 5-formyl group was installed either
Recombinant DNA glycosylase assay
The glycosylase assay was performed in a 10 mL reaction mixture
containing the appropriate reaction buffer (20 mM Tris–HCl at
pH 8, 1 mM DTT, 1 mM EDTA and 0.1 mg mL BSA for eUDG,
ꢂ1
hTDG and hMBD4; 10 mM Bis–Tris–Propane–HCl at pH 7,
2 2 8
by oxidation of the methyl by K S O (ref. 25) or by Pd-catalyzed
ꢂ1
10 mM MgCl
2
, 1 mM DTT and 0.1 mg mL BSA for hSMUG1).
37
Stille coupling reaction with carbon monoxide. The former
reaction resulted in low yield, while the latter one utilized a toxic
organotin reagent as well as CO. In our study, compound 1 was
DNA substrate (50 nM) was incubated with the glycosylase (200
ꢃ
nM) at 37 C in the reaction buffer. The reaction was terminated
by the addition of 5 mL of 100 mM NaOH and an equal volume of
Maxam–Gilbert loading buffer (98% formamide, 10 mM EDTA,
incubated with NaH and nBuLi sequentially followed by treat-
ment with DMF at ꢂ78 ꢃC, leading to the formation of the
ꢂ1
ꢂ1
1
mg mL xylene cyanol and 1 mg mL bromophenol blue)
protected 5foU in 80% yield aer purication (Fig. 1, foU).
When DMF was replaced by dimethyl carbonate, 5caU was ob-
tained under similar conditions with good yield (Fig. 1, caU).
The versatility of our synthetic strategy was further demon-
strated in the preparation of 5hmU. 5-Hydroxymethyl group has
and 50 pmol of an unlabeled ODN as a reannealing competitor.
The backbone was cleaved at the abasic site with NaOH by
ꢃ
heating at 95 C for 30 min. For ODNs containing 5foC or 5foU,
ꢃ
the reactions were stopped by heating to 75 C for 5 min and
then cooled to room temperature. Reaction mixtures were then
resolved on a 15% denaturing polyacrylamide gel. The bands
corresponding to the substrate and product were visualized and
quantied using a Typhoon 9400 phosphorimager (GE Health-
care). Phosphorimager scans were quantied with ImageQuant
TL (GE Healthcare) by generating rectangular boxes around the
substrate or product. Lane-specic background was subtracted
by using analogous rectangles on a portion of the gel lane that
did not correspond to any signal. The percentage conversion
was calculated by dividing the product band intensity by the
sum of the remaining substrate intensity and product intensity.
27,38
routinely been generated by the reduction of 5-formyl group.
The lithiation of compound 1, together with the subsequent
treatment with paraformaldehyde, allowed 5hmU to be created
directly in excellent yield (Fig. 1, hmU). Not surprisingly, the
scope of this method can also be extended to methylation by
methyl iodide to afford T (Fig. 1, T). So far, T, 5hmU, 5foU and
5caU were synthesized from one common ancestor under mild
reaction conditions, by simply changing the electrophiles
accordingly. Furthermore, our strategy potentially provides easy
access to isotopically labeled 5mC derivatives when stable
isotope or radioisotope labeled electrophiles or reagents are
used.
Nuclear extract glycosylase assay
0
The protected 2 -deoxyuridine analogs can be further elabo-
The nuclear extracts from U87 and hES cells were generous gis rated to the corresponding phosphoramidites following the
from Dr Kangling Zhang (University of Texas Medical Branch in standard protocols (Fig. 2): TBDMS deprotection, tritylation of
0
0
Galveston). 5 -End labeled duplex (50 nM) was incubated with the 5 -hydroxyl group, and nally coupling with 2-cyanoethyl
ꢂ1
0
0
nuclear extract (0.5 mg mL ) in a buffer containing 20 mM N,N,N ,N -tetraisopropylphosphorodiamidite (see ESI† for more
Tris–HCl at pH 8, 1 mM DTT, 1 mM EDTA and 0.1 mg mL BSA details).
ꢂ1
39
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0
ꢃ
Fig. 1 Synthesis of 5-methyl-2 -deoxycytidine and derivatives via 5-iodo-2 -deoxyuridine. (a) (1) NaH, THF; (2) nBuLi, then DMF, ꢂ78 C (80%);
ꢃ
ꢃ
(
b) (1) NaH, THF; (2) nBuLi, then dimethyl carbonate, ꢂ78 C (71%); (c) (1) NaH, THF; (2) nBuLi, then paraformaldehyde, ꢂ78 C (86%); (d) (1) NaH,
ꢃ
THF; (2) nBuLi, then MeI, ꢂ78 C (93%); (e) (1) TEA, DMAP, TPSCl, CH
3 4 2
CN; (2) 25% NH OH (90%); (f) Ac O, DMF (92%); (g) (1) TEA, DMAP, TPSCl,
CH
Ac
3
CN; (2) 25% NH
O, DMF (93%).
4
2 3 4
OH (72%); (h) Ac O, DMF (88%); (i) TBAF, THF (85%); (j) ref. 29; (k) (1) TEA, DMAP, TPSCl, CH CN; (2) 25% NH OH (85%); (l)
2
0
With the substituted 2 -deoxyuridine derivatives readily consecutive desilylation, tritylation and phosphitylation
0
available, easy access to 2 -deoxycytidine analogs became sequence. The formation of 5hmC was slightly different: the
40,41
possible via in situ amination.
For example, the uracil in TBDMS-protected 5hmU was desilylated rst, and the 5hmC
protected 5foU was transformed into cytosine with 2,4,6-triiso- phosphoramidite was then generated as described previously
42
propylbenzenesulfonyl chloride (TPSCl) in the presence of (Fig. 1, hmC). Several phosphoramidites mentioned above are
triethylamine (TEA) and N,N-dimethylaminopyridine (DMAP) also commercially available. We selected 5-carboxy-dC-CE phos-
followed by NH OH treatment. Subsequently, the 4-amino group phoramidite from Glen Research and 5-cadC-CE phosphor-
4
was acetylated to allow further modications (Fig. 1, foC). Using amidite from our study for comparison. The 0.25 g (0.276 mmol)
the same method, 5caC and 5mC were obtained from 5caU and package at Glen is priced at $1200. Our synthesis includes 7 steps
0
T, respectively (Fig. 1, caC and mC). For 5foC, 5caC and 5mC, the starting from 5-iodo-2 -deoxyurdine. The overall cost to prepare
corresponding phosphoramidites were synthesized by the 0.276 mmol 5cadC-CE phosphoramidite is less than $250
0
Fig. 2 Standard protocols to synthesize the phosphoramidites of 5-substituted-2 -deoxyuridine analogs. (a) TBAF, THF (77–92%); (b) DMTrCl,
3
pyridine (79–92%); (c) DIPEA, tetrazole in CH CN, 2-cyanoethyl tetraisopropyl phosphoramidite (88–98%).
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considering all the chemicals, solvents and disposables. Our nucleosides and related phosphoramidites, which paves the
strategy is straightforward, cost-friendly with synthetic easiness. way for a better understanding of the epigenetic reprogram-
Compound 1 acted as a common precursor for eight ming and DNA repair pathways.
0
different pyrimidine 2 -deoxynucleosides involved in the active
demethylation pathway. In the rst round, nucleophilic
substitution at 5-position gave rise to four 2 -deoxyuridine
0
Synthesis of ODNs containing modied C or U
analogs. In the second round, 4-position was modied to gain ODNs containing modied pyrimidine nucleobases at a specic
0
access to the 2 -deoxycytidine derivatives. Most of the steps were location in a short sequence were prepared using standard
43,44
under mild conditions with good to excellent yields. The solid-phase DNA synthesis.
The sequence is shown in
divergent synthesis described in this article greatly expanded Fig. 3A. Special attention was given to the deprotection of ODNs
our capacity in the production of epigenetically important containing labile functionalities. In 5caC and 5caU
Fig. 3 Substrate selectivity of recombinant UDGs. (A) Sequences of the synthetic ODNs used for the glycosylase assay; (B and C). Electrophoretic
assays of hTDG (B) and hMBD4 (C) activity on the synthetic ODNs confirmed by autoradiography. The recombinant glycosylase was incubated
32
ꢃ
with P end-labeled duplex DNA at 37 C, followed by alkaline-induced quenching and cleavage of the DNA at glycosylase-generated abasic
sites. S: substrate; P: product.
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phosphoramidites, the carboxylate exists in the methyl ester fragment that can be visualized on a denaturing polyacrylamide
form. The cleavage and deprotection were carried out in 0.4 M DNA gel with a phosphorimager. As expected, U:G pair was well
methanolic NaOH for 17 h at room temperature before being recognized by all four UDGs examined (Fig. 3B, C and S1†), and
neutralized with acetic acid. The formyl group in both 5foC and it was the only mismatch targeted by eUDG under our assay
5
foU phosphoramidites was not protected. It was compatible conditions. On the contrary, 5foC:G and 5caC:G mispairs were
with the mild deprotection condition (0.1 M K CO in only acted upon by hTDG, consistent with the previous
methanol/water) and the standard deprotection treatment with reports.
NH OH at room temperature for an extended period of time. All matched Us in DNA duplexes, removing U, T, 5hmU, 5foU and
the synthetic ODNs were puried by HPLC, and their purities 5caU to different extents (Fig. 3B and Table 1). TDG has a strong
2
3
11,46
Furthermore, hTDG was more active towards mis-
4
3
3
51–53
were examined by MALDI-TOF (Table S1†). The free base preference for bases paired up with G and are followed by G,
composition was veried by HPLC following enzymatic diges- suggesting a pivotal role of TDG in the active demethylation
34
tion, or by GC/MS following acid hydrolysis and conversion to pathway since 5mC is frequently formed in the CpG islands.
35
the t-butyldimethylsilyl ethers.
Similar substrate scopes were observed for hSMUG1 and
hMBD4: hSMUG1 targeted U and its analogs except for T
(Fig. S1C†), while hMBD4 acted on all the U analogs with
a preference for 5foU:G pair (Fig. 3C).
Elucidation of substrate preference of recombinant DNA
glycosylases using synthetic ODNs
In the second set of experiments, the aforementioned 5mC
analogs were paired opposite adenine (A). eUDG acted exclu-
sively on U:A pair (Fig. S1A†). hTDG showed rapid excision of
The last decade has witnessed an ever-increasing interest in
epigenetic editing, especially the reversible DNA modications.
Emerging evidence suggests that uracil DNA glycosylases
5foC from 5foC:A mismatch, and also removed 5caC and U to
(UDGs) play critical roles in the removal of 5mC and related
lesser extents (Fig. 3B). hSMUG1, when rst discovered, was
pyrimidines and thus inuence a broad spectrum of cellular
events such as transcription silencing, cell differentiation and
development. There have been sporadic reports on individual
UDG's action on a limited number of cytosine modica-
54
thought to only have activity on single-stranded DNA (ssDNA).
Recent studies suggest that the substrate scope of this glyco-
12,55–59
sylase can be extended to double-stranded DNA (dsDNA).
Indeed, hSMUG1 demonstrated functional group recognition in
the U:A, 5hmU:A, 5foU:A and 5caU:A pairs, similar to the trend
observed in the C/U:G series (Fig. S1C†). Despite the broad
substrate tolerance in the rst series, hMBD4 failed to remove
any mismatched bases in the C/U:A series (Fig. 3C).
10,11,45–50
tions.
However, a comprehensive analysis of the
substrate selectivity and cleavage efficiency of the UDG super-
family is still not available. Aer securing the panel of synthetic
ODNs bearing ten different cytosine derivatives, we set out to
survey the activity of the UDG family members including E. coli
uracil DNA glycosylase (eUDG), human thymine DNA glyco-
sylase (hTDG), human single-stranded selective monofunc-
tional uracil DNA glycosylase (hSMUG1) and human methyl-
CpG binding domain protein 4 (hMBD4). In the rst series of
experiments, the C or U analogs were paired up with a guanine
The UDGs use a base-ipping mechanism for substrate
recognition: the target base in the duplex is ipped into the
active site prior to the hydrolytic cleavage of the N-glycosidic
60,61
bond.
The substrate specicity is mainly governed by steric
and electronic factors, as well as the stability of the DNA duplex.
Among all the UDGs examined, eUDG exhibited the narrowest
substrate range by removing only the U residue in the mispairs.
Structural analysis suggests that the presence of Tyr66 in the
binding pocket of eUDG excludes sterically bulky 5-substituted
(
G) in the complementary strand to form the duplex. The duplex
3
2
0
was then P-labeled at the 5 -end followed by incubation with
puried recombinant UDG. Finally, the alkaline strand cleavage
3
2
would lead to the formation of a P-labeled short DNA
Table 1 Substrate preference of uracil DNA glycosylases
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62
pyrimidines. hTDG, on the other hand, is considered a versa- greater signicance because emerging data suggest that it is
tile DNA glycosylase by targeting a wide range of 5mC analogs. operational during cellular differentiation and in normal stem
These modied bases can be accommodated in the hTDG cells, but is uniformly defective in human cancer cells poten-
46
63,64
binding pocket via p–p stacking interaction with Tyr152.
tially due to defects in formation as well as processing.
With
Additionally, a network of hydrogen bond donors and acceptors the array of above-mentioned synthetic ODNs, it is possible to
46
provides greater exibility in the binding site. Moreover, the interrogate the DNA glycosylase activity in cell extracts from
substrate discrimination of hTDG can be explained by the both cancer cells and normal stem cells. First, UDG activity in
electronic inductive property of the 5-substituents. Formyl HeLa nuclear extract was probed using the panel of duplexes
group, an electron-withdrawing substituent, is expected to containing the C/U:G pairs (Fig. 4A and S2A†). Substantial
increase the acidity of the cleaved base to increase excision activities against U:G, 5hmU:G and 5foU:G were detected. It
activity. Methyl and hydroxymethyl substituted bases are rela- appeared that HeLa nuclear extract lacks signicant hTDG
tively poorer substrates due to their electron-donating effects. activity. The observed excision of 5hmU and 5foU was likely due
6
5
Unlike hTDG, hSMUG1 does not exploit the electronic effects of to hSMUG1 activity, in agreement with a previous study.
12
the substituents. Rather, it relies on the duplex stability for
hTDG activity is known to be regulated transcriptionally by
substrate discrimination. A previous study indicates that both promoter methylation and by tumor suppressor protein
12
49
66,67
hMBD4 specically recognizes T and 5hmU. Our study p53.
The mRNA and protein levels of hTDG can be stimu-
expanded its substrate repertoire to include U, 5foU and 5caU, lated in a p53-dependent fashion. It has been reported that
68
but only when they are opposite G. The lack of detectable gly- HeLa cells lack detectable levels of p53 protein. The absence of
cosylase activity against the C/U:A pairs requires further p53 results in the downregulation of hTDG. Furthermore, it was
mechanistic and structural analysis.
reported that high levels of hTDG causes cell cycle arrest in the
S-phase in several human cell lines, including HeLa cells.
69
SUMOylation-mediated degradation warrants an hTDG-free
progression until the G2-phase. The downregulation of hTDG
could contribute to the abundant transition mutations at
methylated CpG sites frequently observed in human cancer
cells. Our synthetic ODNs, together with the bioassay, were
powerful tools in identifying a defective component of the
demethylation pathway in HeLa cells.
Probing DNA glycosylase activity in cell extracts using
synthetic ODNs
The processing of 5mC and its derivatives is believed to involve
either enzymatic oxidation or deamination followed by glyco-
sylase removal and DNA repair, indicating a potentially
complicated intersection of DNA damage repair with epigenetic
reprogramming. This pathway has taken on substantially
Fig. 4 DNA glycosylase activity in cell extracts. The glycosylase activity in the nuclear extract (NE) from HeLa (A), U87 (B) and hES (C) cells to
32
generate alkaline-sensitive sites was assayed using P end-labeled duplex DNAs.
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This screening strategy was then expanded to human U87
glioblastoma cells and human embryonic stem (hES) cells
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(
Fig. 4B, C, S2B and S2C†). U87 cells, similar to HeLa cells,
demonstrated robust glycosylase activity against U, 5hmU and
foU, suggesting a common mechanism underlying the epige-
5
netic regulation of cell proliferation in these cell lines. hES cells,
on the other hand, exhibited dramatically different excision
patterns. In addition to several U analogs, hES nuclear extract
was also able to remove 5foC efficiently and cleave 5caC to
a much lesser extent (Fig. 4C), consistent with the substrate
preference of hTDG. Recent studies indicate that mouse
embryonic stem (mES) cells have high levels of 5hmC, signi-
cantly lower level of 5foC and almost undetectable levels of
2
3,70
5caC.
Furthermore, the nuclear extract of mES cells is known
10
to harbor 5caC-selective TDG activity. DNA methylation is
considered an important epigenetic regulation of pluripotency
and differentiation of stem cells. Previous results and our own
data suggest that BER mediated by TDG plays critical roles in
8 E. L. Fritz and F. N. Papavasiliou, Genes Dev., 2010, 24, 2107–
2114.
maintaining
development.
epigenetic
stability
during
embryonic
9 S. Cortellino, J. Xu, M. Sannai, R. Moore, E. Caretti,
A. Cigliano, M. Le Coz, K. Devarajan, A. Wessels,
D. Soprano, L. K. Abramowitz, M. S. Bartolomei,
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A. J. Klein-Szanto, Y. Matsumoto, D. Kobi, I. Davidson,
C. Alberti, L. Larue and A. Bellacosa, Cell, 2011, 146, 67–79.
71
Conclusions
In summary, we report the divergent synthesis of phosphor-
amidites carrying epigenetically important 5mC analogs from
one common precursor. Nucleophilic substitutions at 5-posi-
1
0 Y. F. He, B. Z. Li, Z. Li, P. Liu, Y. Wang, Q. Tang, J. Ding,
Y. Jia, Z. Chen, L. Li, Y. Sun, X. Li, Q. Dai, C. X. Song,
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1 A. Maiti and A. C. Drohat, J. Biol. Chem., 2011, 286, 35334–
0
tion of 5-iodo-2 -deoxyuridine furbished a set of U derivatives
1
1
including T, 5hmU, 5foU and 5caU. In situ amination then
converted the U series to C analogs. Our synthetic strategy
provided unprecedented easiness and yields in generating these
important DNA building blocks. The modied pyrimidines were
incorporated into 24 mer ODNs in a site-specic manner using
solid-support DNA synthesis. This battery of ODNs allowed the
interrogation of substrate preference and excision efficiency of
a family of recombinant UDGs. We further envisaged the eval-
uation of DNA glycosylase activity in human cell extracts using
these synthetic ODNs, giving attention to the cancer cells as well
as ES cells. Given the importance of epigenetic reprogramming
in cell development, differentiation and disease progression,
the synthetic methods and chemical probes we developed will
be widely utilized for a better understanding of this intriguing
biological event.
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
2
2 Z. Vanikova, M. Janouskova, M. Kambova, L. Krasny and
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This work was supported in part by start-up funding from VCU
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