FEBS Letters 582 (2008) 1629–1633

Effect of 1-methyladenine on double-helical DNA structures

Hao Yang, Yingqian Zhan, Dickson Fenn, Lai Man Chi, Sik Lok Lam*

Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Received 6 March 2008; revised 9 April 2008; accepted 13 April 2008

Available online 22 April 2008

Edited by Hans Eklund

Abstract Methylation at the N1 site of adenine leads to the for-mation of cytotoxic 1-methyladenine (m1A). Since the N1 site ofadenine is involved in the hydrogen bonding of TÆA and AÆT Wat-son–Crick base pairs, it is expected that the pairing interactionswill be disrupted upon 1-methylation. In this study, high-resolu-tion NMR investigations were performed to determine the effectof m1A on double-helical DNA structures. Interestingly, insteadof disrupting hydrogen bonding, we found that 1-methylation al-tered the TÆA Watson–Crick base pair to T(anti)Æm1A(syn) Hoo-gsteen base pair, providing insights into the observed differencesin AlkB-repair efficiency between dsDNA and ssDNA.� 2008 Federation of European Biochemical Societies.Published by Elsevier B.V. All rights reserved.

Keywords: NMR; DNA; 1-Methylation; Adenine; Hoogsteenbase pair

1. Introduction

DNA methylation is an essential process for normal devel-

opment and functioning of organisms which involves the cova-

lent addition of a methyl group to a nucleobase. Traditionally,

it has only been considered as cytosine 5-methylation in the

context of the CpG dinucleotide [1,2] that control gene expres-

sion [3], cause genomic imprinting [4] and abolish the immune

response induced by the CpG motifs flanked by two purines

and two pyrimidines in the 5 0 and 3 0 directions, respectively

[5]. Owing to the presence of abundant alkylating agents and

various mutagenic agents, DNA in living organisms is vulner-

able to alkylation and this methylation process sometimes be-

comes detrimental to living organisms.

Besides the C5 site of cytosine, methylation can take place at

different positions such as N- and O-sites of nucleobases.

Among them, N- and O-methylated bases have been found

to be more cytotoxic [6–8]. Methylation at the N1 site of ade-

nine leads to the formation of 1-methyadenine (m1A) which

blocks DNA replication if not repaired [9,10]. Owing to the

Abbreviations: m1A, 1-methyladenine; m6A, N6-methyladenine; NMR,nuclear magnetic resonance; dsDNA, double-stranded DNA; ssDNA,single-stranded DNA; PAGE, polyacrylamide gel electrophoresis; 1D,one-dimensional; 2D, two-dimensional; NOE, nuclear Overhausereffect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, totalcorrelation spectroscopy; DQF, double-quantum-filtered; COSY, cor-relation spectroscopy; HSQC, heteronuclear single quantum coherencespectroscopy; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid;WATERGATE, water suppression by gradient-tailored excitation

*Corresponding author. Fax: +852 2603 5057.E-mail address: lams@cuhk.edu.hk (S.L. Lam).

0014-5793/$34.00 � 2008 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2008.04.013

fact that the N1 site of adenine is involved in the hydrogen

bonding of TÆA and AÆT Watson–Crick base pairs, it is ex-

pected that the base pair structure of DNA double-helix will

be seriously affected upon 1-methylation. In order to maintain

the genetic stability and integrity of DNA, appropriate repair

of the damaged DNA is crucial to avoid mutations and lethal

diseases such as cancers [1,11,12].

Recently, a DNA damage reversal enzyme, AlkB, which di-

rectly mediates an oxidative demethylation of m1A and N3-

methylcytosine, has been found to suppress both genotoxicity

and mutagenesis, providing a new DNA repair pathway

[1,10,11,13–16]. Although AlkB demethylates m1A both in sin-

gle-stranded DNA (ssDNA) and double-stranded DNA

(dsDNA), the repair in dsDNA is 10-fold less efficient than

that in ssDNA [11,14]. Kinetic studies have also shown a pref-

erence of AlkB-repair on ssDNA substrate over its dsDNA

counterpart [17]. Although extensive work has been carried

out to study DNA methylation, the effect of N-methylation

on DNA structures and dynamics remain elusive.

In this study, we have investigated the TÆA base pair struc-

ture upon methylation at the N1 site of adenine using high-res-

olution nuclear magnetic resonance (NMR) spectroscopy. We

found that upon 1-methylation on adenine, the TÆA Watson–

Crick base pair is switched to TÆm1A Hoogsteen base pair.

The structural findings provide possible explanations for the

observed differences in the AlkB-repair efficiency between

dsDNA and ssDNA.

2. Materials and methods

2.1. Sample designTwo 17-nt DNA samples were designed to contain either a TÆm1A or

TÆA base pair in the middle of the double-helical stem regions and theywere named as ‘‘Tm1A-oligo’’ and ‘‘TA-oligo’’, respectively (Fig. 1A).In order to simplify the sample preparative work, a 5 0-GAA loop wasadded to one of the terminals to connect the two strands of the double-helix.

2.2. Sample preparationAll DNA samples were synthesized using an Applied Biosystems

model 392 DNA synthesizer and purified using denaturing polyacryl-amide gel electrophoresis (PAGE) and diethylaminoethyl Sephacel an-ion exchange column chromatography. For incorporating an m1A intoTm1A-oligo, 1-methyl deoxyadenosine phosphoramidite (ChemGenesInc.) was used and the base deprotection step was performed at 37 �Cfor 16 h. Owing to the necessary use of concentrated ammoniumhydroxide in the deprotection step, m1A was partially converted toN6-methyladenine (m6A) via a base-catalyzed Dimorth rearrangement[10,18]. By limiting the sample quantity loaded onto the gel, the m1Aand m6A species were successfully separated using PAGE (AppendixA, S1). NMR samples were prepared by dissolving 0.5 lmol of purifiedDNA samples into 500 ll of buffer solution containing 150 mM so-dium chloride, 10 mM sodium phosphate (pH 7.0), and 0.1 mM 2,2-di-methyl-2-silapentane-5-sulfonic acid (DSS).

blished by Elsevier B.V. All rights reserved.

Fig. 1. (A) Sequence design and (B) imino 1H spectra at 5 �C ofTm1A-oligo (left) and TA-oligo (right).

1630 H. Yang et al. / FEBS Letters 582 (2008) 1629–1633

2.3. NMR studyAll NMR experiments were performed using either a Bruker ARX-

500 or AV-500 spectrometer operating at 500.13 MHz and acquired at25 �C unless stated otherwise. For studying labile proton resonancesignals, the samples were prepared in a 90% H2O/10% D2O buffer solu-tion. One-dimensional (1D) imino spectra were acquired using thewater suppression by gradient-tailored excitation (WATERGATE)pulse sequence [19,20], and two-dimensional (2D) WATERGATE-nu-clear Overhauser effect spectroscopy (NOESY) experiments were per-

Fig. 2. (A) T4H3–m1A14H8 and (B) T4H3–m1A14H62 NOEs were observeSchemes of TÆm1A Hoogsteen and reverse Hoogsteen base pairs. Black arroT4CH3–m1A14H62 NOE was observed in the WATERGATE-NOESY specsegments of the 1D WATERGATE spectrum of Tm1A-oligo at 5 �C. Owihydrogen bonds in Hoogsteen base pair, the linewidth of m1A14H62 is narr

formed with a mixing time of 300 ms. For studying nonlabile protonsignals, the solvent was exchanged with a 100% D2O buffer solution.2D NOESY (100 and 300 ms mixing time), total correlation spectros-copy (TOCSY) (75 ms mixing time) and double-quantum-filtered-cor-relation spectroscopy (DQF-COSY) were performed, and 4K · 512data sets were collected. In general, the acquired data were zero-filledto give 4K · 4K spectra with a cosine window function applied to bothdimensions. For DQF-COSY, a sine window function was used forvicinal proton–proton coupling measurements.

Backbone 31P signals were assigned using 2D TOCSY and 1H– 31Pheteronuclear single quantum coherence spectroscopy (HSQC) experi-ments. The 1H and 31P spectral widths of the HSQC experiment wereset to 10 and 2 ppm, respectively, and 2K · 256 data sets were col-lected. Zero-filling, baseline correction and cosine bell window func-tion were applied to generate a 2K · 2K data matrix. 31P chemicalshifts were indirectly referenced to DSS using the derived nucleus-spe-cific ratio of 0.404808636 [21]. 2D 1H– 31P COSY experiments with aGaussian inversion pulse centered at the H3 0 region were also per-formed and 2K · 128 data sets were collected. The F1 dimension wasextended to 180 complex points by forward linear prediction andzero-filled to 2K complex points for 3J H30P measurements.

3. Results and discussion

In this study, we aimed at investigating the effect of 1-methyl-

ation of adenine on DNA double-helical structure. High-resolu-

d in the WATERGATE-NOESY spectrum of Tm1A-oligo at 5 �C. (C)ws indicate characteristic NOEs across each of the base pairs. (D) Atrum of Tm1A-oligo at 5 �C. The projections on 2D spectra show theng to the reduced exchange rate with the solvent upon formation ofower than that of m1A14H61.

H. Yang et al. / FEBS Letters 582 (2008) 1629–1633 1631

tion NMR spectroscopic investigations focusing on (i) base

pairing mode, (ii) sugar pucker, and (iii) backbone conforma-

tion of the double-helical stem regions of Tm1A-oligo and

TA-oligo were performed. Sequential proton resonance assign-

ments were made by studying the fingerprint regions in 2D

NOESY and WATERGATE-NOESY spectra (Appendix A,

S2 and S3).

3.1. Base pairing mode

CÆG and TÆA Watson–Crick base pairs were formed in the

stem regions of Tm1A-oligo and TA-oligo except the

T4Æm1A14 base pair. This is evidenced by the appearance

and chemical shifts of the stem G and T imino signals (Fig.

1B) as well as the appearance of G imino-C amino and T imi-

no-AH2 nuclear Overhauser effects (NOEs) in their 2D

WATERGATE-NOESY spectra (Appendix A, S2 and S3)

[22]. On the other hand, for the T4Æm1A14 base pair, the T4

imino signal appears more upfield than T and G imino signals

of Watson–Crick base pairs, suggesting T4Æm1A14 adopts a

special base pairing mode. The presence of T4H3–m1A14H8

(Fig. 2A) and T4H3–m1A14H62 NOEs (Fig. 2B) in the

WATERGATE-NOESY spectrum at 5 �C suggest that

T4Æm1A14 is either a Hoogsteen [23] or reverse Hoogsteen base

pair [24] (Fig. 2C).

To differentiate these two base pairing modes, we estimated

the characteristic proton–proton distances in Hoogsteen and

reverse Hoogsteen base pairs. Two nucleic acid crystal struc-

tures containing an AÆT Hoogsteen base pair (PDB ID:

Fig. 3. NOE crosspeaks of (A) H6/H8–H1 0 of T4 and m1A14, and (B) H5–same baseline threshold. 2D NOESY was performed with a mixing time of

1RSB) [25] and an AÆU reverse Hoogsteen base pair (PDB

ID: 1FQZ) [26] were used. Since AÆT reverse Hoogsteen base

pair is not commonly found in antiparallel DNA duplexes,

we made use of an RNA AÆU base pair and added a methyl

group to the C5 position of U using SYBYL 7.3 (Tripos

Inc.) so as to estimate the distances between thymine methyl

protons (CH3) and adenine H-bonded amino proton (H62).

As these distances were found to be 4.3–6.0 and 7.0–7.5 A

in Hoogsteen and reverse Hoogsteen base pairs, respectively,

the presence of T4CH3–m1A14H62 NOE (Fig. 3D) supports

T4Æm1A14 adopts the Hoogsteen pairing mode. In addition,

the observed upfield T4 imino chemical shift is also consistent

with previously observed T imino proton chemical shifts of

Hoogsteen base pair in DNA duplexes [23,27], confirming

T4Æm1A14 is a Hoogsteen base pair.

1-Methylation of adenine also affects the base orientation. In

the stem regions of Tm1A-oligo and TA-oligo, all bases in

Watson–Crick base pairs adopt an anti glycosidic orientation

with respect to their sugar rings as evidenced by their intranu-

cleotide H8/H6–H1 0 NOEs with intensities weaker than that of

cytosine H5–H6 NOEs (Appendix A, S2 and S3) [22]. For

bases in a syn orientation, the intranucleotide H8/H6–H1 0 dis-

tance is �1.5 A shorter than that in the anti orientation and

thus a strong NOE is expected. In the T4Æm1A14 Hoogsteen

base pair, m1A14 possesses a strong H8–H1 0 NOE with inten-

sity comparable to cytosine H5–H6 NOEs (Fig. 3), suggesting

that m1A14 adopts the syn orientation. In addition, the H2 0

chemical shift of m1A14 was found to be more upfield than

H6 of C1, C3, C11, C13 and C16. These NOEs were plotted using the100 ms.

1632 H. Yang et al. / FEBS Letters 582 (2008) 1629–1633

that of A14 (Appendix A, S4 and S5), which is consistent with

the findings in H2 0 chemical shift for the syn orientation [27–

29]. On the contrary, the H6–H1 0 NOE of T4 is relatively weak

(Fig. 3A), suggesting that T4 is in the anti orientation. As a re-

sult, 1-methylation of A14 leads to the formation of T4(anti)-

Æm1A14(syn) Hoogsteen base pair.

3.2. Sugar pucker

The effect of 1-methylation of adenine on the sugar pucker

was determined by extracting the vicinal 3J H10H20 and 3JH10H200

coupling constants from the DQF-COSY spectra at 25 �C.

The percentage of S-state (%S) [30] of each nucleotide was

determined and the %S of all nucleotides before and after 1-

methylation were found to be above 60% (Appendix A, S6).

Apart from T4, which is opposite to m1A14, shows a 22%

reduction in the %S value, the sugar pucker of all other nucle-

otides remain similar, indicating 1-methylation only has a local

effect.

3.3. Backbone conformation

To study the effect of 1-methylation of adenine on the double-

helical backbone conformation, we first examined the 31P chem-

ical shifts as they have been shown to be sensitive to changes in

Fig. 4. 1H– 31P COSY spectra of (A) Tm1A-oligo and (B) TA-oligo.

DNA backbone conformation, especially the a and f torsion an-

gles [31]. The 31P resonance assignments were shown in the 1H–31P COSY spectra (Fig. 4). Excluding the two most upfield sig-

nals that correspond to the phosphates near the loop region, the31P chemical shifts of both Tm1A-oligo and TA-oligo show a

narrow dispersion (�0.7 ppm) (Fig. 4), indicating no abrupt

change in the backbone conformation of the stem regions.

The 3JH3 0P coupling constants were also measured in order

to determine the percentage population of BI conformation

(%BI) [31]. The %BI values of the double-helical stem region

vary slightly within �40–60% (Appendix A, S7), which agrees

well with the narrow dispersion of the observed 31P chemical

shifts in both oligomers. Except for the phosphate between

C13 and A14 (C13p) in which the %BI value increased 18%

upon 1-methylation of A14, the differences in the %BI values

of all other nucleotides between the two oligomers were less

than 7%. This finding was also in agreement with the largest31P chemical shift difference observed in C13p (Fig. 4), indicat-

ing 1-methylation of adenine has only a local effect on the

backbone conformation.

In short, 1-methylation of adenine causes a change of TÆAWatson–Crick base pair in DNA double-helices to TÆm1A

Hoogsteen base pair. Instead of the anti base orientation in

Watson–Crick base pair, m1A adopts a syn orientation in Hoo-

gsteen base pair. Although m1A has a local effect on both the

sugar pucker and backbone conformation, 1-methylation of

adenine does not disrupt base pairing but just switches the base

pairing mode. The formation of Hoogsteen base pair retains the

TÆm1A base pairing and stacking within the double-helix,

which possibly makes the recognition and thus the repair of

m1A lesion in dsDNA by AlkB less efficient than that in

ssDNA [14]. In order to examine if relationship exists between

base pair structures and mutagenicity of N-methylated base le-

sions, we are in the progress to determine the structural features

of other N-methylated base lesions. Further structural findings

will enhance our understandings of the DNA repair process.

Acknowledgement: The work described in this paper was fully sup-ported by a grant from the Research Grants Council of the HongKong Special Administrative Region (Project No. CUHK401105).

Appendix A. Supplementary data

Preparative-scale PAGE picture of Tm1A-oligo, NOESY

spectra showing the sequential resonance assignments of

Tm1A-oligo and TA-oligo, tables summarizing proton chemi-

cal shifts, 3JH10H20 ,3JH10H200 and 3JH30P results of Tm1A-oligo

and TA-oligo. Supplementary data associated with this article

can be found, in the online version, at doi:10.1016/j.febslet.

2008.04.013.

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