Structure of (5′ S)-8,5′-cyclo-2′-deoxyguanosine in DNA

Article abstract of DOI:10.1021/ja207407n

Diastereomeric 8,5′-cyclopurine 2′-deoxynucleosides, containing a covalent bond between the deoxyribose and the purine base, represent an important class of DNA damage induced by ionizing radiation. The 8,5′-cyclo-2′-deoxyguanosine lesion (cdG) has been r

Full text of DOI:10.1021/ja207407n

ARTICLE
pubs.acs.org/JACS
Structure of (50S)-8,50-Cyclo-20-deoxyguanosine in DNA
Hai Huang,† Rajat S. Das,‡ Ashis K. Basu,‡ and Michael P. Stone*,†
†Department of Chemistry, Center in Molecular Toxicology, Center for Structural Biology, and the Vanderbilt-Ingram Cancer Center,
Vanderbilt University, Nashville, Tennessee 37235, United States
‡Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States
S Supporting Information
b
ABSTRACT:
Diastereomeric 8,50-cyclopurine 20-deoxynucleosides, containing a covalent bond between the deoxyribose and the purine base,
represent an important class of DNA damage induced by ionizing radiation. The 8,50-cyclo-20-deoxyguanosine lesion (cdG) has
been recently reported to be a strong block of replication and highly mutagenic in Escherichia coli. The 8,50-cyclopurine-
20-deoxyriboses are suspected to play a role in the etiology of neurodegeneration in xeroderma pigmentosum patients. These lesions
cannot be repaired by base excision repair, but they are substrates for nucleotide excision repair. The structure of an
oligodeoxynucleotide duplex containing a site-specific S-cdG lesion placed opposite dC in the complementary strand was obtained
by molecular dynamics calculations restrained by distance and dihedral angle restraints obtained from NMR spectroscopy. The
S-cdG deoxyribose exhibited the O40-exo (west) pseudorotation. Significant perturbations were observed for the β, γ, and χ torsion
angles of the S-cdG nucleoside. WatsonꢀCrick base pairing was conserved at the S-cdG dC pair. However, the O40-exo
3
pseudorotation of the S-cdG deoxyribose perturbed the helical twist and base pair stacking at the lesion site and the 50-neighbor
dC dG base pair. Thermodynamic destabilization of the duplex measured by UV melting experiments correlated with base stacking
3
and structural perturbations involving the modified S-cdG dC and 30- neighbor dT dA base pairs. These perturbations may be
3
3
responsible for both the genotoxicity of this lesion and its ability to be recognized by nucleotide excision repair.
’ INTRODUCTION
in xeroderma pigmentosum complementation group C (XP-C)
patients.24 They are also believed to play roles in Cockayne
syndrome,21 breast and ovarian cancer,22 and familial Medi-
terranean fever.25
Hydroxyl radicals cause a variety of damage in DNA, affecting
the nucleobases1 or deoxyribose sugars,2 or both,3 as in the
case of tandem 8,50-cyclopurine 20-deoxynucleoside lesions.4 At
20-deoxyguanosines in DNA, hydrogen abstraction by a hydroxyl
radical at the C50 position of the deoxyribose followed by attack
at the C8 carbon of guanine forms an N7-centered radical, which
may be oxidized to produce diastereomeric 8,50-cyclo-20-deoxy-
guanosines (cdG).4ꢀ10 The corresponding 8,50-cyclo-20-deoxy-
adenosines (cdA) have also been characterized.3,4,7,9ꢀ15 For both
cdG and cdA, the diastereomeric ratio at the C50 position depends
on experimental conditions and DNA conformation.5ꢀ7,13,16ꢀ18
The 8,50-cyclopurine-20-deoxynucleosides are believed to be
important contributors to the genetic toxicology of oxidative
stress and inflammation.4 They have been detected at the
nucleotide level,5,11 in DNA,5,19ꢀ21 and in cells in vitro,6 in
human urine,18 and in vivo.21ꢀ23 The formation of 8,50-cyclopur-
ine-20- deoxynucleosides might contribute to neurologic disease
In Escherichia coli, S-cdG is a block to DNA replication, is
highly mutagenic, and is refractory to repair.26 It induced 34%
mutations upon induction of the SOS response. Most mutations
were S-cdG f A mutations, though S-cdG f T mutations
and deletions of the 50-neighbor dC at a low level also were
observed.26 It has been reported in a preliminary study that
S-cdG does not block primer elongation by Klenow DNA
polymerases, and dATP is preferentially incorporated opposite
the lesion.27
Computational studies predicted that the incorporation of the
cdA stereoisomers into DNA would result in helical distortions at
Received: August 16, 2011
Published: November 21, 2011
r
2011 American Chemical Society
20357
dx.doi.org/10.1021/ja207407n J. Am. Chem. Soc. 2011, 133, 20357–20368
|

Journal of the American Chemical Society
ARTICLE
Scheme 1. Numbering Scheme of the Oligodeoxynucleotide
Duplex Containing the (50S)-8,50-Cyclo-20-deoxyguanosine
(S-cdG) 50-Nucleotide
the lesion site.28ꢀ30 Both the R- and S-diastereomers of the 8,50-cA
ribonucleoside have been crystallized.31,32 Both exhibited the anti
0
0
conformation about the glycosyl bond with χO4 ꢀC1 ꢀN9ꢀC8
=
29.8° or 27.4°, respectively. The fused six-member ring
C8ꢀN9ꢀC10ꢀO40ꢀC40ꢀC50 adopted the half-chair conforma-
tion with the O40 and C40 out of plane. The deoxyribose adopted
the O40-exo (0T1) pseudorotation with P = ∼289° and τm = ∼48°.
Molecular mechanics calculations predicted that the cdA diaseter-
eomers maintain the O40-exo pseudorotation when placed oppo-
site dT in DNA.28 The NMR data and ab initio calculations suggest
that incorporation of the S-cdA into di- or trinucleotides does not
change the O40-exo deoxyribose pseudorotation.33
Herein, we report the structure of the S-cdG dC pair in
3
50-d(GTGCXTGTTTGT)-30 50-d(ACAAACACGCAC)-30, con-
3
taining the DNA sequence of p53 codons 272ꢀ275, where
X denotes the S-cdG (Scheme 1). The lesion is located in codon
273. The S-cdG remains stacked into the duplex and participates
in WatsonꢀCrick hydrogen bonding with the complementary
dC. However, the S-cdG deoxyribose shifts to the O40-exo
pseudorotation with P = 280°. This alters the γ and δ backbone
torsion angles. Additionally, the β and χ torsion angles are
changed from those in B-DNA. The twist and base pair shift
helicoidal parameters are perturbed at the C4 G21 and X5 C20
3
3
base pairs. The purine ring is anti about the glycosyl bond, and
the fused six-membered ring adopts the half-chair conformation
with O40 and C40 out of plane.
Figure 1. NOE connectivity of base H8/H6 protons with deoxyribose
H10 protons of the S-cdG containing duplex: (A) modified strand;
(B) complementary strand.
’ RESULTS
Synthesis and Characterization of the S-cdG-Modified
Oligodeoxynucleotide. The S-cdG-modified oligodeoxy-
nucleotide 50-GTGCXTGTTTGT-30, containing the sequence
of p53 codons 272ꢀ275 in which the lesion was located in codon
273, was synthesized by a modification of the method reported by
Romieu (Scheme S1 in the Supporting Information).34 The syn-
thesis of N2-isobutyryl-50-phenylthio-20,50-dideoxyguanosine gave
70% yield from N2-isobutyryl-20-deoxyguanosine. The yield was
improved to 91% when the exocyclic N2-dG amino group was
protected with DMF. However, the DMF protection was unstable
in the subsequent NaBH4 reduction step. Therefore, after cycliza-
tion and TBDMS protection of the 30-hydroxyl group, it was
replaced with an isobutyryl group. The modified oligodeoxynucleo-
tide was synthesized using solid phase phosphoramidite chemistry
and characterized by HPLC and MALDI-TOF mass spectrometry.
NMR Resonance Assignments. The nonexchangeable pro-
tons of the S-cdG-modified duplex were assigned on the basis of
the sequential connectivity of the base proton H6 or H8 dipolar
couplings with H10 deoxyribose protons (Figure 1).35,36 For the
modified strand, the NOE sequential connectivity was observed
from G1 to C4. Since the S-cdG nucleotide lacked a proton at the
C8 carbon, the sequential connectivity exhibited an interruption
at X5. However, the X5 H10 proton was identified at 6.14 ppm; it
exhibited a weak X5 H10 f T6 H6 NOE, suggesting that the
distance between these two protons was greater than that in
B-DNA. The sequential NOE connectivity resumed from T6 to
T12. For the modified strand, all of the deoxyibose H10 protons
were observed within a narrow chemical shift window, between
5.8 and 6.3 ppm. The complete sequential NOE connectivity was
observed for the complementary strand.
The assignments of X5 deoxyribose protons were made by
analysis of scalar and dipolar couplings. Figure 2 displays a tile
plot derived from a NOESY spectrum obtained at 60 ms mixing
time. X5 H10 exhibited strong dipolar couplings with H20 and
H200; weak scalar couplings were also observed. H30 exhibited
strong dipolar couplings with H20, H200, and H40, whereas the
20358
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
scalar couplings were unobservable. H40 exhibited both scalar
and dipolar couplings with the single H50 proton. The geminal
H20 and H200 protons were assigned from their NOEs to H10 and
H30. H20 exhibited a weaker NOE with H10 than did H200,
whereas it exhibited a stronger NOE with H30 than did H200.
In B-DNA, H200 resonances are usually more downfield than
H20 resonances. However, the X5 H20 resonance was observed at
2.55 ppm, whereas the H200 resonance was observed at 2.27 ppm.
For the remainder of the duplex, the H20, H200, H30, and H40
deoxyribose resonances were assigned unequivocally. The reso-
nance assignments of the nonexchangeable DNA protons are
tabulated in Table S1 of the Supporting Information.
connectivity was observed from G1 f T2 f G3 f G21 to X5, and
from G7 f T8 f T9 f T10 to G11. At and adjacent to the lesion
site, G21 N1H exhibited NOEs with C4 N4H1 and N4H2, and X5
N1H exhibited NOEs with the complementary C20 N4H1 and
N4H2. At the 30-neighbor base pair, the T6 N3H resonance was
not observed, but A19 H2 exhibited NOEs to both X5 N1H and
G7 N1H, suggesting A19 was still intercalated. Except for the
terminal base pairs, the remaining NOE cross-peaks arising from
WatsonꢀCrick hydrogen bonding were observed.
Deoxyribose Coupling Constants. Figure 4 displays the
expansion of an ECOSY spectrum38 in the region of deoxyribose
The resonances of the base imino protons were assigned on
the basis of sequential connectivity in NOESY spectra, and the
assignments were supported by NOEs to the amino protons of
WatsonꢀCrick base pairs (Figure 3).37 The NOE sequential
Figure 4. Expansion of the ECOSY spectrum used for the measurement
of 3JH1 ꢀH2 and 3JH1 ꢀH2 coupling constants. Except for X5, G11, and
T12, all H200 protons exhibited greater chemical shifts than H20 protons.
The geminal H20 and H200 protons of G11 and T12 were not resolved, and
X5 H200 was upfield from H20.
0
0
0
00
Figure 2. Tile plot derived from a NOESY spectrum obtained at a
mixing time of 60 ms showing the assignment of S-cdG nonexchange-
able protons.
Figure 3. Assignment of the base imino and amino protons on the basis of the NOE connectivity. The NOE interactions of the imino protons with the
opposite base arising from WatsonꢀCrick base pairing are labeled as follows: (a) X5 N1H f C20 N4H2; (b) X5 N1H f A19 H2; (c) X5 N1H f C20
N4H1; (d) G7 N1H f C18 N4H2; (e) G7 N1H f A19 H2; (f) G7 N1H f A17 H2; (g) G7 N1H f C18 N4H1; (h) G11 N1H f C14 N4H2; (i) G11 N1H f
C14 N4H1; (j) G3 N1H f C22 N4H2; (k) G3 N1H f C22 N4H1; (l) G21 N1H f C4 N4H2; (m) G21 N1H f C4 N4H1; (n) T2 N3H f A23 H2;
(o) T10 N3H f A15 H2; (p) T9 N3H f A16 H2; and (q) T8 N3H f A17 H2.
20359
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
3
3
H10 correlations with H20 and H200. The JH1 ꢀH2 and JH1 ꢀH2
2.6 and 7.0 Hz, respectively. Consistently, the H10ꢀH20 cross-
0
0
0
00
3
peak was weak. The 3JH4 ꢀH5 was 5.4 Hz, whereas the JH3 ꢀH4
0
0
0
0
coupling constants were measured from the multiplicities of
the cross-peaks. The 3JH1 ꢀH2 and JH1 ꢀH2 values for X were
was not measurable. Except for the terminal nucleotides, the
3
5
0
0
0
00
3
0
0
JH1 ꢀH2 values for all other nucleotides were 8ꢀ10 Hz, and the
3
0
00
JH1 ꢀH2 values were 5ꢀ7 Hz, suggesting that the deoxyriboses
3
adopted C10-exo or C20-endo conformations. The J coupling
constants for the deoxyribose protons are tabulated in Table S2
of the Supporting Information.
Phosphodiester Backbone Conformation. The 31P reso-
nances were assigned from a 31PꢀH30 HMBC spectrum (Figure
S1 in the Supporting Information). Except for X5, each phos-
phodiester exhibited a heteronuclear coupling with H30 of the
50-neighbor. Figure 5 displays the 31P NMR of the S-cdG con-
taining duplex compared with the corresponding unmodified
duplex. At the modified nucleotide, the 31P resonance shifted up-
field, indicating a backbone perturbation at the modified base.
The other 31P resonances were clustered within a modest
chemical shift range, centered in the spectral region characteristic
of B DNA.
Chemical Shift Perturbations. Chemical shifts of the non-
exchangeable protons between the S-cdG containing duplex
and the corresponding unmodified duplex were compared
(Figure 6). Significant changes were observed at X5 and the 50-
and 30-neighboring nucleotides of the modified strand. C4 H6,
H10, and H200 shifted downfield by 0.21, 0.38, and 0.99 ppm,
Figure 5. 31P NMR of the S-cdG containing duplex compared with the
corresponding unmodified duplex: (A) unmodified duplex; (B) S-cdG
containing duplex.
Figure 6. Proton chemical shift perturbations of the S-cdG containing duplex compared with the unmodified duplex: (A) base protons of the S-cdG-
modified strand; (B) deoxyribose protons of the S-cdG-modified strand; (C) base protons of the complementary strand; (D) deoxyribose protons of the
complementary strand.
20360
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
Figure 7. 1H NMR of the S-cdG containing duplex compared with the corresponding unmodified duplex at different temperatures: (A) unmodified
duplex; (B) S-cdG containing duplex.
respectively; X5 H200 shifted upfield by 0.55 ppm; and T6 H6,
CH3, and H10 shifted downfield by 0.36, 0.31, and 0.22 ppm,
respectively. In contrast, the chemical shift perturbations for the
complementary strand were small, with the exception of A19 H20,
which shifted upfield by 0.21 ppm.
NMR data, which suggested that structural perturbations for the
duplex were localized at and adjacent to the lesion site. No base
pair distance restraints were used for the T6 A19 base pair, and no
3
torsion angle restraints were used for the C4 G21, X5 C20, and
3
3
T6 A19 base pairs. The restraints used for the structure refine-
3
ment are summarized in Table 1.
Thermal Stability of the S-cdG Modified Duplex. The
thermal melting of the modified duplex containing the S-cdG
was monitored using UV spectroscopy in 100 mM NaCl at pH
7.0. It exhibited a melting temperature (Tm) of 46 ( 1 °C, as
compared to the unmodified DNA that exhibited a Tm of 55 °C.
Thus, the incorporation of S-cdG reduced the Tm by 9 °C.
The rMD calculations for the S-cdG containing duplex were
performed from the initial A- and B-form starting structures. Ten
final structures, five each for the A- and B-DNA starting struc-
tures, with lowest energies, were obtained. All structures con-
verged as indicated by pairwise rmsd comparisons (Table 1). The
accuracies of the emergent structures were evaluated by com-
parison of theoretical NOE intensities calculated by CORMA40
for the refined structure to the experimental NOE intensities
1
Figure 7 displays H NMR spectra of the S-cdG containing
duplex and the corresponding unmodified duplex at dif-
ferent temperatures. In the modified duplex, the X5 imino
resonance exhibited significantly more line broadening at
45 °C than the corresponding G5 imino resonance of the
unmodified duplex. For the modified duplex, the T6 N3H
resonance was not observed at 5 °C, suggesting that the S-cdG
to yield sixth root residuals (R1 ).41 The overall residuals, as
x
well as the residuals for intra- or internucleotide NOEs, were
x
consistently less than 0.1 (Table 1). R1 values for each nucleotide
were less than 0.15 (Figure S2 in the Supporting Information).
Thus, the refined structures provided accurate depictions of the
NOE data.
nucleotide also significantly perturbed the 30-flanking T6 A19
3
base pair.
Structural Refinement. A total of 426 distance restraints,
including 274 intranucleotide and 152 internucleotide restraints,
were calculated from the intensities of NOE cross-peaks using
Structure of the S-cdG-Containing Duplex. The significant
perturbations involved the modified strand. Figure 8 shows an
expanded view at the lesion site. The S-cdG nucleotide was in the
O40-exo, “west” pseudorotation (Figure 9B), with P = 280.2° and
τm = 47.6°. The heavy atoms N9, O30, and C50 were axial about
the deoxyribose ring. With the exception of the terminal nucleo-
tides, all other deoxyribose pseudorotations were either C10-exo
or C20-endo. Figure 9A displays the six-membered ring
C8ꢀN9ꢀC10ꢀO40ꢀC40ꢀC50 conformation. It adopted the
MARDIGRAS (Table S3 in the Supporting Information).39
A
total of 29 NOEs involving the S-cdG protons were used as
restraints. A total of 45 empirical distance restraints arising from
WatsonꢀCrick base pairing interactions were used, as were 165
empirical torsion angle restraints that were applied to refine the
nonterminal nucleotides. These were justified on the basis of the
20361
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
Table 1. rMD Restraints and Statistical Analysis of rMD
Converged Structures of the S-cdG Containing Duplex
total restraints for rMD calculation
experimental NOE distance restraints
636
426
274
152
29
intranucleotide NOE restraints
internucleotide NOE restraints
NOEs of S-cdG
empirical base pairing restraints
empirical torsion angle restraints
backbone torsion angles restraints
deoxyribose torsion angles restraints
45
165
95
70
Structure Statisticsa
NMR R-factor (R1 ) (ꢁ10ꢀ2
)
5.75
4.66
7.97
0.55
x
Figure 9. Ring conformations of the S-cdG in the refined structure:
(A) six-member ring C8ꢀN9ꢀC10ꢀO40ꢀC40ꢀC50; (B) 20-deoxyribose.
intranucleotide NOEs
internucleotide NOEs
rmsd deviation of refined structures
a The mixing time used to calculate R1 was 250 ms. Rx1
=
x
∑|(a0)i1/6 ꢀ (ac)i1/6|/|(a0)1i /6|, where (a0) and (ac) are the intensities
of observed (nonzero) and calculated NOE cross-peaks, respectively.
Figure 10. Base pairing and base stacking of the refined structure of the
S-cdG containing duplex at the lesion site. The pink arrows indicate
anticipated hydrogen bonding interactions. (A) C4 G21 and X5 C20
3
3
base pairs. (B) X5
C A
20 and T6 19 base pairs.
3
3
Figure 10 shows the base stacking and base pairing at the
lesion site. The 50-neighbor C4 G21 base pair exhibited a shift
Figure 8. Expanded views of the refined structure of the S-cdG contain-
ing duplex at the lesion site: (A) view from the major groove; (B) view
from the minor groove.
3
of ꢀ1.0 Å, resulting in the displacement of C4 toward the major
groove. At the C4 f X5 step, an increased twist of 49° with
respect to the X5 C20 base pair was evident. In contrast, the
3
helix was underwound at the X5 f T6 step. Additionally, the
envelope (half boat) conformation. Helicoidal analysis of the
backbone torsion angles showed that, at the lesion site, the
β (PꢀO50ꢀC50ꢀC40) angle shifted from the characteristic
∼180° to ꢀ87°. The γ (O50ꢀC50ꢀC40ꢀC30) angle shifted
from ∼50° to ꢀ67°. Modest perturbations of the δ (C50ꢀ
C40ꢀC30ꢀO30) and ζ (C30ꢀO30ꢀPꢀO50) torsion angles were
also observed from ∼120° to +149° and from ∼ ꢀ90° to ꢀ59°,
respectively. There was also a modest change for the glycosyl
torsion angle χ from ∼ ꢀ120° to ꢀ157°. C4 H200 was proximate
to the X5 purine ring. In contrast, X5 H200 was farther from the
X5 purine ring compared to the H200 protons in B DNA.
30-neighbor base pair T6 A19 exhibited a greater than normal
3
base pair opening of ꢀ11.3°.
Molecular Dynamics Calculations in Explicit Solvent. A
molecular dynamics simulation was carried out in explicit water
at constant pressure at 300 K, for 5 ns. The distances of the atoms
involving in the WatsonꢀCrick hydrogen bonding were mea-
sured in the trajectories. Figure 11 shows the distances of guanine
N1H f cytosine N3 and the thymine N3H f adenine N1 of
some base pairs observed in the trajectories. During this simula-
tion, no changes in the monitored distances were observed for
20362
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
Figure 11. Distances of guanine N1H f cytosine N3 and the thymine N3H f adenine N1 of some base pairs in the trajectories of the molecular
dynamics simulations conducted in explicit solvent at 300 K: (A) T2 A23 base pair; (B) C4 G21 base pair; (C) X5 C20 base pair; (D) T6 A19 base pair;
3
3
3
3
(E) T8
A A
17 base pair; (F) T9 16 base pair.
3
3
the G C and C G base pairs including the damaged X5 C20 base
Although the repair of S-cdG by the human NER system remains
to be determined, Jasti et al.26 demonstrated that in DNA the
S-cdG lesion was incised by the UvrABC nuclease of E. coli. The
covalent bond between C8 of guanine and C50 of the deoxyribose
in the 8,50-cyclo-20-deoxyguanosine locks the modified nucleo-
tide in the anti conformation. This is believed to hinder the
flipping of the purine ring from the duplex, which is consistent
with the observation that the 8,50-cyclopurine-20-deoxynucleo-
sides are not repaired by BER.42,43 If not repaired, the S-cdG
lesion is mutagenic. In SOS-induced E. coli, a mutation frequency
of 34% was observed. Most mutations were S-cdG f A muta-
tions, though S-cdG f T mutation and a deletion of the
50-neighbor C also was observed.26 Hence, it was of interest to
determine the structure of S-cdG in DNA.
3
3
3
pair. In contrast, at the T6 A19 base pair, an opening occurred at
3
∼0.9 ns, as indicated by the distances of T6 O2 f A19 H2, T6
N3H f A19 N1, and T6 O4 f A19 N6H1 jumping from ∼3.5 Å to
∼5.5 Å, from ∼2.0 Å to ∼3.5 Å, and from ∼1.8 Å to ∼2.2 Å,
respectively. Other nonterminus T A base pairs exhibited no
3
remarkable changes.
’ DISCUSSION
Interest in the 8,50-cyclopurine-20-deoxynucleoside lesions has
been piqued by evidence that in mammalian cells 8,50-cyclo-
20-deoxyadenosine (cdA) diastereomers3,4,7,9ꢀ15 are repaired by
nucleotide excision repair (NER),42,43 an idea that was suggested
earlier,5,6 and not by base excision repair. Also, the bacterial DNA
N-glycosylases endo III and FpG do not excise S-cdG from DNA,
suggesting that, like cdA, it also is a substrate for NER.27
Structure of S-cdG in DNA. The present study reveals that
S-cdG remains stacked into the duplex and participates in
WatsonꢀCrick hydrogen bonding with the complementary
20363
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
dC. However, the S-cdG deoxyribose shifts to the O40-exo
pseudorotation, as opposed to the “south” pseudorotation
(C20-endo) observed in B-DNA or the “north” pseudorotation
(C30-endo) in A-DNA.44,45 This corroborates computational
studies on 8,50-cyclopurine-20-deoxynucleosides.28 Crystal struc-
tures of the cA ribonucleoside also exhibited the O40-exo
pseudorotation,31,32 and an NMR and DFT study of di- and
trideoxynucleotides containing S-cdA indicated the O40-exo
deoxyribose.33 The O40-exo pseudorotation introduces signifi-
cant helicoidal perturbation into the modified strand of DNA.
This involves changes in the S-cdG phosphodiester backbone
torsion angles β (PꢀO50ꢀC50ꢀC40), γ (O50ꢀC50ꢀC40ꢀC30),
δ (C50ꢀC40ꢀC30ꢀO30), and ζ (C30ꢀO30ꢀPꢀO50) from
∼180° to ꢀ87°, from ∼50° to ꢀ67°, from ∼120° to 149°,
and from ∼ ꢀ90° to ꢀ59°, respectively. These changes perturb
of the altered ζ backbone torsion angle of S-cdG, which results in
the opening of the 30-neighbor base pair. While the MD simula-
tions occur on a different time scale than the NMR experiments,
in the MD simulations, transient opening of the T6 A19 base pair
3
is predicted (Figure 11). In contrast, the thermal melting
experiments (Figure 7) suggest that the 50-neighbor C4 G21
3
base pair is more stable with respect to imino proton exchange.
StructureꢀActivity Relationships. a. DNA Repair. In hu-
man global genome NER, the XPC/HR23B complex51ꢀ55 is
believed to be involved in damage recognition. The XPA protein
is also essential for NER. Yang et al.56 reported that it exists as a
homodimer either in the free state or as a complex with RPA. For
example, it binds to mismatched bubble substrates, including the
C8-dG adducts of AF, AAF, and 1-nitropyrene, and the T[6,4]T
photoproducts.57 XPA is proposed to be involved in the verifica-
tion of DNA damage.54,55 It may also recruit repair factors and
stabilize repair intermediates, since it binds more efficiently to
undamaged ds-ssDNA junctions with ssDNA branches,57 inter-
mediate structures found in NER.
the helicoidal twist and base pair shift parameters at the C4 G21
3
and X5 C20 base pairs from ∼30° to 49° and from ∼0 Å to
3
ꢀ1.0 Å, respectively. These changes are consistent with the
upfield shift of the 31P resonance at S-cdG. These conclusions
also are consistent with computational studies, which predict that
the O40-exo pseudorotation of the cdA deoxyribose should alter
The destabilization of the S-cdG modified duplex and the
perturbation of the X5 C20 and T6 A19 base pairs is likely
3
3
the helical twist parameter for the modified cdA dT base pair as
3
relevant with respect to NER. Thermal destabilization of the
duplex is believed to modulate recognition of a diverse group of
damages by XPC.54,58ꢀ61 From studies of the yeast XPC
orthologue Rad4 bound to DNA containing a cyclobutane
pyrimidine dimer, Min and Pavletich62 concluded that Rad4
may exploit the destabilization of two base pairs. Interestingly,
the 5R-thymine glycol lesion, another substrate for NER, also
destabilizes two base pairs in DNA.63 The perturbation of the
X5 C20 and T6 A19 base pairs in the S-cdG modified duplex may
compared to the flanking base pairs.28 In addition, the modified
cdA dT base pair exhibited an altered base pair shift parameter.
3
The altered ζ backbone torsion angle of S-cdG (ꢀ59°) results
in the greater than normal base opening of ꢀ11.3° for the
30-neigbhor T6 A19 base pair (Figure S4 in the Supporting
3
Information). Additionally, the glycosyl torsion angle χ
(O40ꢀC10ꢀN9ꢀC2) is modified from ∼ ꢀ120° to ꢀ157°.
This places the six-member ring C8ꢀN9ꢀC10ꢀO40ꢀC40ꢀC50
into the half-boat conformation. The bond between X5 C8 and
C50 pulls X5 H40 and H50 closer to the purine ring as compared to
the H40, and H500 protons in B-DNA. This is consistent with the
downfield chemical shifts of both X5 H40 and H50. In contrast, X5
H200 is farther from the X5 purine ring compared to the H200
protons in B-DNA, consistent with its upfield shift compared to
that in the unmodified duplex.
3
3
facilitate extrusion of both C20 and A19 (but not X5) out of the
helix, enabling XPC/HR23B to recognize S-cdG prior to
recruiting XPA.
b. Error-Prone Replication Bypass. The bond between C8 of
guanine and C50 of 20-deoxyribose locks the N-glycosyl torsion
angle of S-cdG in the anti domain. Therefore, during translesion
synthesis, an incoming dCTP can form a WatsonꢀCrick base
pair, whereas an incoming dTTP might form a wobble pair. The
insertion of both dATP and dTTP were noted in pol V-dependent
TLS by Jasti et al.26 Significantly, they noted the genotoxicity of
the S-cdG lesion, which implied that DNA polymerases have
difficulty in bypassing this locked nucleotide. They speculated
that accommodation of the S-cdG lesion within the active site of
the polymerase likely involves rotational adjustments of the
nucleoside around the glycosyl bond.26 Thus, future structural
Thermodynamic Considerations. Energetically, the O40-exo
pseudorotation is disfavored due to the axial orientation of all
substituent heavy atoms.28 The helical perturbation of the
modified strand associated with the unusual O40-exo deoxyribose
at the lesion site is consistent with the 9 °C decrease in the Tm of
the modified duplex as compared to the unmodified control. The
destabilization likely involves structural perturbations observed
for the modified X5 C20 and 30-neighbor T6 A19 base pairs, and
3
3
accompanying base stacking perturbations. Indeed, the X5 imino
resonance exhibits increased line broadening at 45 °C as
compared to the G5 imino resonance of the unmodified duplex
(Figure 7). Exchange-mediated line broadening of DNA imino
protons is normally associated with the formation of an open
state of the base pair in which the imino proton is freed from its
hydrogen bond and is accessible to the base that catalyzes the
proton exchange,46ꢀ50 but S-cdG is locked in the anti conforma-
tion about the glycosyl bond and incapable of flipping out of the
duplex. It seems possible that if the complementary nucleotide
studies of template primers containing the S-cdG lesion com-
3
plexed with error-prone polymerase will be of interest.
’ CONCLUSIONS
The structure of S-cdG has been determined when placed
opposite dC in DNA. The S-cdG dC and the flanking base pairs
3
maintain WatsonꢀCrick hydrogen bonding. However, S-cdG
exhibits the O40-exo deoxyribose pseudorotation in DNA. This
introduces significant helicoidal and base stacking perturbations
C
20 nucleotide flips out, this might facilitate proton exchange by
allowing water to enter the duplex to access the X5 imino proton,
but more detailed studies of the exchange kinetics of the X5 and
neighboring imino protons are warranted.49,50 For the modified
duplex, the T6 N3H resonance is not observed, suggesting
increased exchange with solvent for the imino proton of the 30-
into the duplex. The imino proton of the 30-neighbor T A base
3
pair undergoes increased exchange with solvent, whereas the
50-neighbor C G base pair is only moderately influenced.
3
Collectively, these structural and thermodynamic perturbations
may be important in modulating the recognition of the S-cdG
lesion during nucleotide excision repair.
flanking T6 A19 base pair (Figure 7). This may be a consequence
3
20364
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
’ EXPERIMENTAL SECTION
This product was added to 50 mL of methanol, and NaBH4 (0.174 g,
4.6 mmol) was added to it in three portions. This reaction mixture was
stirred at room temperature for 1 h, and the reaction was monitored by
TLC (93/7 CHCl3/MeOH, v/v). The excess borohydride was neutra-
lized by addition of 1 N HCl dropwise to the solution. The solution was
passed through a Celite pad and evaporated to dryness. The resulting
yellow solid was purified by silica gel column chromatography with a
step gradient of methanol (0ꢀ5%) in chloroform. The product was
isolated as a white solid (0.37 g, yield of 36%).
Synthesis. a. N2-((Dimethylamino)methylene)-20-deoxyguanosine
(1). To a suspension of 20-deoxyguanosine (10 g, 35.06 mmol) in dry
methanol (100 mL) was added N,N-dimethylformamide dimethyl acetal
(18.7 mL, 140.24 mmol) dropwise with vigorous stirring. The mixture
was stirred at room temperature under argon for 72 h. The solid product
was isolated by filtration, washed with cold methanol, and dried. The
product was isolated as a white solid in quantitative yield.
b. N2-DMF-50-phenylthio-20,50-dideoxyguanosine (2). N2-DMF-
20-deoxyguanosine (1) (1 g, 3.74 mmol) and diphenyl disulfide (1.63 g,
7.48 mmol) were dissolved in 15 mL of dry DMF under argon, PBu3
(1.85 mL, 7.48 mmol) was slowly added dropwise, and the mixture was
stirred at room temperature for 6 h. The reaction was monitored by thin
layer chromatography (TLC) (90/10 CH2Cl2/MeOH, v/v). The reac-
tion was quenched with 10 mL of water and evaporated to a glassy
syrupy residue. It was purified by silica gel column chromatography with
a step gradient of methanol (0ꢀ7%) in DCM as the mobile phase. The
product was isolated as a white foam (1.42 g, yield of 91%).
g.
(50S)-N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-50-O-(4,40-
dimethoxytrityl)-50,8-cyclo-20-deoxyguanosine (8). (50S)-N2-Isobutyryl-
30-O-(tert-butyldimethylsilyl)-50,8-cyclo-20-deoxyguanosine (6) (0.4 g,
0.89 mmol) was dissolved in 3 mL of dry pyridine and evaporated to
dryness. This process was repeated twice. The residual white solid was
dissolved in 10 mL of dry pyridine and DMT-Cl (0.92 g, 2.7 mmol), and a
few crystals of DMAP were added to it. The mixture was heated at 80 °C
and stirred under nitrogen atmosphere for 8 h. It was monitored by TLC
(94/5/1 CHCl3/MeOH/NEt3, v/v). The solution was cooled to ∼5 °C in
an ice bath, and reaction was quenched with methanol. The solvents were
removed under reduced pressure, and the resulting yellow solid was
purified by silica gel column chromatography with a step gradient of
methanol (0ꢀ1%) in chloroform containing 1% TEA. The product was
isolated as a white solid (0.39 g, yield of 58%).
c. N2-DMF-50,8-cyclo-20,50-dideoxyguanosine (3). Previously crushed
N2-DMF-50,8-cyclo-20,50-dideoxyguanosine (2) (1.4 g, 3.38 mmol) and
triethyl phosphate were added to an argon-purged 2 L quartz reactor and
dissolved in 1 L of dry acetonitrile via sonication. This solution was degassed
by bubbling argon for 40 min. The reactor was sealed under argon
atmosphere and irradiated at 254 nm UV light for 20 h. The reaction was
monitored using TLC (85/15 CHCl3/MeOH, v/v). The solution was
evaporated to dryness, and the resulting brownish yellow solid was purified
by silica gel column chromatography with a step gradient of methanol
(0ꢀ10%) in CHCl3. The product was isolated as a light yellowish white
solid (0.53 g, yield of 52%).
h. (50S)-N2-Isobutyryl-50-O-(4,40-dimethoxytrityl)-50,8-cyclo-20-
deoxyguanosine. (50S)-N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-
50-O-(4,40-dimethoxytrityl)-50,8-cyclo-20-deoxyguanosine (7) (0.3 g,
0.40 mmol) was dissolved in 15 mL of dry THF, and a solution of 1 M
TBAF in THF (0.8 mL, 0.8 mmol) was added. The reaction was stirred
under nitrogen atmosphere for 5 h and monitored by TLC (92/7/1
CHCl3/MeOH/NEt3, v/v). The solvents were removed under
reduced pressure, and the resulting yellow solid was purified by silica
gel column chromatography with a step gradient of methanol (0ꢀ2%)
in chloroform containing 1% TEA. The product was isolated as a white
solid (0.24 g, yield of 95%).
d. N2-DMF-30-O-(tert-butyldimethylsilyl)-50,8-cyclo-20,50-dideoxy-
guanosine (4). N2-DMF-50,8-cyclo-20,50-dideoxyguanosine (3) (1.4 g,
4.6 mmol) and imidazole (1.27 g, 18.7 mmol) were dried and dissolved
in 20 mL of dry DMF. TBDMS-Cl (1.39 g, 9.2 mmol) was added to
this solution while stirring under nitrogen atmosphere. The reaction
mixture was stirred at room temperature for 20 h and monitored by TLC
(93/7 CHCl3/MeOH, v/v). The solvent was dried under nitrogen, and
the resulting semisolid was purified via silica gel column chromato-
graphy with a step gradient of methanol (0ꢀ3%) in chloroform. The
product was isolated as a white solid (1.35 g, yield of 70%).
i. (50S)-50,8-Cyclo-20-deoxyguanosine Phosphoramidite Derivative.
(50S)-N2-Isobutyryl-50-O-(4,40-dimethoxytrityl)-50,8-cyclo-20-deoxy-
guanosine (from the previous reaction) (0.092 g, 0.14 mmol) was
dissolved in dry dichloromethane and evaporated to dryness. This
process was repeated twice. The solid was dissolved in 5 mL of dry
dichloromethane and kept under argon. Diisopropylethylamine (51
μL, 0.29 mmol) was added to it, then 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (34 μL, 0.15 mmol) was added to the stirring
solution dropwise. The reaction was checked by TLC (95/4/1
CHCl3/MeOH/NEt3, v/v). After 1 h, the solution was cooled to
∼5 °C with an ice bath, 51 μL of DIEA and 0.2 mL of methanol were
added to it. The solvents were removed under reduced pressure, and
the resulting light yellow semisolid was purified twice via silica gel
column chromatography with a step gradient of methanol (0ꢀ1%) in
chloroform containing 1% TEA. The product was isolated as a white
solid (0.084 g, yield of 70%).
Oligodeoxynucleotides. The 50-d(GTGCGTGTTTGT)-30 and
50-d(ACAAACACGCAC)-30 were synthesized and purified by anion-
exchange chromatography by the Midland Certified Reagent Co.
(Midland, TX). The dodecamer containing the S-cdG 50-d(GTG-
CXTGTTTGT)-30, where X represents the S-cdG, was synthesized,
purified, and characterized using a slightly amended procedure of the
synthesis reported by Romieu et al.34 The purity of the modified
oligodeoxynucleotide was assessed by HPLC and mass spectrometry.
Oligodeoxynucleotides were desalted by chromatography on Sephadex
G-25. The 50-d(GTGCGTGTTTGT)-30 or 50-d(GTGCXTGTTTGT)-30
was annealed with the complementary strand 50-d(ACAAACACG-
CAC)-30 in buffer containing 10 mM NaH2PO4, 100 mM NaCl, and
50 μM Na2EDTA (pH 7.0), respectively. The resulting duplexes were
heated to 95 °C for 10 min and cooled to room temperature. They were
e. N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-50,8-cyclo-20,50-
dideoxyguanosine (5). N2-DMF-30-O-(tert-butyldimethylsilyl)-50,8-
cyclo-20,50-dideoxyguanosine (4) (1.1 g, 2.63 mmol) was dissolved in
a mixture of 50 mL of methanol and 10 mL of 29% aqueous ammonia,
and the reaction mixture was stirred overnight at room temperature. The
solvents were removed under reduced pressure. The resulting white
powder was coevaporated with 5 mL of dry pyridine three times. This
white solid was dissolved in 12 mL of dry pyridine, a few crystals of
DMAP were added to it, and isobutyryl chloride (0.56 mL, 5.26 mmol)
was added to it dropwise under nitrogen atmosphere. This reaction
mixture was stirred at room temperature for 8 h and monitored by TLC
(93/7 CHCl3/MeOH, v/v). The solvent was dried under reduced
pressure, and the resulting yellow solid was purified by silica gel column
chromatography with a step gradient of methanol (0ꢀ2%) in DCM. The
product was isolated as a white solid (1.0 g, yield of 88%).
f. (50S)-N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-50,8-cyclo-20-
deoxyguanosine (7). N2-Isobutyryl-30-O-(tert-butyldimethylsilyl)-50,8-
cyclo-20,50-dideoxyguanosine (5) (1.0 g, 2.3 mmol) was dissolved in
250 mL of dry 1,4-dioxane, SeO2 (1.28 g, 11.5 mmol), and the mixture
was refluxed for 24 h. The reaction was monitored by TLC (93/7
CHCl3/MeOH, v/v). The hot solution was passed through a Celite pad
and washed with 20 mL of 10% methanol in chloroform. The filtrate was
dried under reduced pressure to produce a brownish white powder of
N2-isobutyryl-30-O-(tert-butyldimethylsilyl)-50-oxo-50,8-cyclo-20-deoxyguanosine.
20365
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
purified by DNA grade hydroxylapatite chromatography using a gradient
from 10 to 200 mM NaH2PO4 in 100 mM NaCl, 50 μM Na2EDTA (pH
7.0), and desalted using Sephadex G-25.
Melting Temperature. Melting temperatures of the DNA du-
plexes were measured in 10 mM NaH2PO4, 100 mM NaCl, 50 μM
EDTA (pH 7.0) by UV/vis spectroscopy at 260 nm. The strand
concentration was 10 μM. The thermal scan proceeded from 10 to
80 °C with an interval of 1 °C. The melting temperatures were calculated
by differentiating the absorbance profiles.
Molecular Dynamics Calculations. Restrained molecular
dynamics (rMD) calculations for the modified oligodeoxynucleotide
duplexes utilized a simulated annealing approach.76 The partial charges
on the cdG nucleotide (Figure S5 in the Supporting Information) were
obtained from density function theory (DFT) calculations using a
neutral total charge, utilizing the B3LYP/6-31G* basis set and the
program GAUSSIAN.77 To obtain the starting structures used for
rMD calculations, the cdG-modified duplex was energy minimized using
200 iterations with the conjugate gradients algorithm. The rMD
calculations were conducted with AMBER78 and the parm99 force field.
The generalized Born (GB) model79 with parameters developed by Tsui
and Case80 was used for implicit water simulation. The program
CORMA was utilized to calculate the NOE intensities from the
structures emergent from rMD calculations.
Molecular dynamics simulations in explicit water were performed
using the AMBER force field. The average structure converged from the
simulated annealing rMD calculations was used as the starting structure.
This was placed in an 8.0 Å cubic TIP3P water box in each direction.81
The necessary Na+ ions were added to neutralize the duplex. The system
was subjected to 1000 iterations of potential energy minimization using
steepest descents. The solvent was brought to thermal equilibrium by a
MD simulation at constant volume for 10,000 iterations with an
integrator time of 1 fs, at 300 K. After equilibration of the system at
300 K, MD calculations were performed at constant pressure for 5 ns
with an integrator time of 1 fs. Bond lengths involving hydrogens were
fixed with the SHAKE algorithm.82 The particle mesh Ewald (PME)
method was used to approximate nonbonded interactions.83,84 The
cutoff radius for nonbonded interactions was 8.0 Å. The PTRAJ program
from the AMBER package was used to analyze the MD trajectories. Helicoidal
analyses were carried out with the programs CURVES85 and 3DNA.86
NMR. Samples were at 1.0 mM strand concentration. Samples for the
nonexchangeable protons were dissolved in 500 μL in 10 mM NaH2-
PO4, 100 mM NaCl, 50 μM Na2EDTA (pH 7.0). They were exchanged
with D2O and suspended in 280 μL of 99.996% D2O. The pH was
adjusted with dilute DCl or NaOD. Experiments were performed at
800 MHz. COSY and NOESY spectra were recorded with 512 real data
in the t1 dimension and 2048 real data in the t2 dimension. NOESY
spectra were zero-filled during processing to create a matrix of 1024 ꢁ
1024 real points. NOESY experiments used TPPI quadrature
detection64 and mixing times of 60, 150, 200, and 250 ms. The relaxation
delay was 1.5 s. The TOCSY mixing time was 80 ms. The temperature
was 25 °C. Chemical shifts were referenced to water. Data were
processed using TOPSPIN65 and analyzed with the program SPARKY.66
The ECOSY data were recorded with 1024 real data in the t1 dimension
and 4096 real data in the t2 dimension.38 The spectrum was zero-filled
during the process to create a matrix of 2048 ꢁ 16384 to increase digital
resolution. The temperature was 30 °C. Samples for the observation of
exchangeable protons were dissolved in 500 μL of 10 mM NaH2PO4,
100 mM NaCl, 50 μM EDTA, (pH 7.0) containing 9:1 H2O/D2O (v/v)
(pH 7.0). Experiments were performed at 500 MHz. The temperature
was 5 °C. The Watergate sequence was used for water suppression.67
The mixing time was 250 ms. The 31PꢀH1 experiments were carried out
1
31
at the H frequency 600 MHz. PꢀH30 3J couplings were applied to
determine the phosphodiester backbone conformation.68 31P chemical
shifts were referenced using indirect shift ratios.69
’ ASSOCIATED CONTENT
S
Supporting Information. Complete ref 77; Scheme S1,
b
synthesis of the phosphoramidite of S-cdG; Table S1, 1H
chemical shifts of the nonexchangeable protons of the S-cdG
modified duplex; Table S2, J coupling constants and deoxy-
Distance and Dihedral Angle Restraints. Footprints were
drawn around NOE crosspeaks obtained at a mixing time of 250 ms.
Their intensities were determined by volume integrations. These were
combined as necessary with intensities generated from complete relaxa-
tion matrix analysis of a starting structure to generate a hybrid NOE
intensity matrix.41,70 The program MARDIGRAS39,40,71 iteratively
refined the hybrid intensity matrix and optimized agreement between
calculated and experimental NOE intensities. The RANDMARDI
algorithm39 carried out iterations, randomizing peak volumes within
limits specified by the input noise level.71 Calculations were initiated
using isotropic correlation times of 2, 3, and 4 ns. Analysis of these data
yielded experimental distance restraints used in rMD calculations (Table S3
in the Supporting Information) and the corresponding standard deviations
for the distance restraints.
3
ribose pseudorotation of the S-cdG modified duplex; Table S3,
NOE distance restraints for the S-cdG modified duplex; and
31
Figure S1, PꢀH30 HMBC spectrum of the S-cdG modified
duplex; Figure S2, nucleotide-by-nucleotide sixth root residuals
for the average refined structure of the S-cdG modified duplex;
Figure S3, backbone torsion angles of the refined structure of the
S-cdG-containing duplex; Figure S4, base pairing and base
stacking helicoidal parameters of the S-cdG-containing duplex;
and Figure S5, calculated electrostatic potentials for the S-cdG
nucleotide. This material is available free of charge via the
Internet at http://pubs.acs.org.
The deoxyribose pseudorotational angles (P) were estimated by
3
examining the JHH of sugar protons.72 The data were fit to curves
’ AUTHOR INFORMATION
relating the coupling constants to the pseudorotation (P), the sugar
pucker amplitude (ϕ), and the percentage S-type conformation. The
pseudorotation and amplitude ranges were converted to the five dihedral
Corresponding Author
michael.p.stone@vanderbilt.edu
1
31
angles ν0 to ν4. Coupling constants measured from Hꢀ P HMBC
spectra were applied73,74 to the Karplus relationship75 to determine the
backbone dihedral angle ε (C40ꢀC30ꢀO30ꢀP), related to the
H30ꢀC30ꢀO30ꢀP angle by a 120° shift. The ζ (C30ꢀO30ꢀPꢀO50)
backbone angles were calculated from the correlation between ε and ζ in
B-DNA.68 Empirical restraints preserved WatsonꢀCrick hydrogen
bonding and prevented propeller twisting between base pairs, except
’ ACKNOWLEDGMENT
This work was supported by NIH Grants CA-55678 (M.P.S.)
and ES-013324 (A.K.B.).
’ REFERENCES
(1) Cooke, M. S.; Evans, M. D.; Dizdaroglu, M.; Lunec, J. FASEB J.
2003, 17, 1195–1214.
for the A6
T
19 base pair. Except for the modified, the flanking, and the
3
terminal base pairs, other backbone torsion angle restraints were using
empirical data derived from B-DNA.44
(2) Dedon, P. C. Chem. Res. Toxicol. 2008, 21, 206–219.
20366
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
(41) James, T. L. Curr. Opin. Struct. Biol. 1991, 1, 1042–1053.
(42) Kuraoka, I.; Bender, C.; Romieu, A.; Cadet, J.; Wood, R. D.;
Lindahl, T. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3832–3837.
(43) Brooks, P. J.; Wise, D. S.; Berry, D. A.; Kosmoski, J. V.;
Smerdon, M. J.; Somers, R. L.; Mackie, H.; Spoonde, A. Y.; Ackerman,
E. J.; Coleman, K.; Tarone, R. E.; Robbins, J. H. J. Biol. Chem. 2000,
275, 22355–22362.
(3) Keck, K. Z. Naturforsch., B 1968, 23, 1034–1043.
(4) Jaruga, P.; Dizdaroglu, M. DNA Repair 2008, 7, 1413–1425.
(5) Dizdaroglu, M. Biochem. J. 1986, 238, 247–254.
(6) Dizdaroglu, M.; Dirksen, M. L.; Jiang, H. X.; Robbins, J. H.
Biochem. J. 1987, 241, 929–932.
(7) Dirksen, M. L.; Blakely, W. F.; Holwitt, E.; Dizdaroglu, M. Int. J.
Radiat. Biol. 1988, 54, 195–204.
(8) Chatgilialoglu, C.; Bazzanini, R.; Jimenez, L. B.; Miranda, M. A.
Chem. Res. Toxicol. 2007, 20, 1820–1824.
(44) Arnott, S.; Hukins, D. W. L. Biochem. Biophys. Res. Commun.
1972, 47, 1504–1509.
(9) Boussicault, F.; Kaloudis, P.; Caminal, C.; Mulazzani, Q. G.;
Chatgilialoglu, C. J. Am. Chem. Soc. 2008, 130, 8377–8385.
(10) Chatgilialoglu, C.; Ferreri, C.; Terzidis, M. A. Chem. Soc. Rev.
2011, 40, 1368–1382.
(11) Mariaggi, N.; Cadet, J.; Teoule, R. Tetrahedron 1976, 32, 2385–
2387.
(45) Saenger, W. In Principles of Nucleic Acid Structure; Springer:
New York, 1984.
(46) Teitelbaum, H.; Englander, S. W. J. Mol. Biol. 1975, 92, 79–92.
(47) Mandal, C.; Kallenbach, N. R.; Englander, S. W. J. Mol. Biol.
1979, 135, 391–411.
(48) Englander, S. W.; Kallenbach, N. R. Q. Rev. Biophys. 1984,
(12) Raleigh, J. A.; Kremers, W.; Whitehouse, R. Radiat. Res. 1976,
65, 414–422.
(13) Fuciarelli, A. F.; Shum, F. Y.; Raleigh, J. A. Biochem. Biophys. Res.
16, 521–655.
(49) Leroy, J. L.; Kochoyan, M.; Huynh-Dinh, T.; Gueron, M. J. Mol.
Biol. 1988, 200, 223–238.
Commun. 1986, 134, 883–887.
(14) Chatgilialoglu, C.; Guerra, M.; Mulazzani, Q. G. J. Am. Chem.
Soc. 2003, 125, 3839–3848.
(15) Navacchia, M. L.; Chatgilialoglu, C.; Montevecchi, P. C. J. Org.
Chem. 2006, 71, 4445–4452.
(16) Jaruga, P.; Theruvathu, J.; Dizdaroglu, M.; Brooks, P. J. Nucleic
Acids Res. 2004, 32, e87.
(17) Belmadoui, N.; Boussicault, F.; Guerra, M.; Ravanat, J. L.;
Chatgilialoglu, C.; Cadet, J. Org. Biomol. Chem. 2010, 8, 3211–3219.
(18) Jaruga, P.; Dizdaroglu, M. Biochem. Biophys. Res. Commun.
2010, 397, 48–52.
(50) Folta-Stogniew, E.; Russu, I. M. Biochemistry 1996, 35, 8439–
8449.
(51) Legerski, R.; Peterson, C. Nature 1992, 359, 70–73.
(52) Masutani, C.; Sugasawa, K.; Yanagisawa, J.; Sonoyama, T.; Ui,
M.; Enomoto, T.; Takio, K.; Tanaka, K.; van der Spek, P. J.; Bootsma, D.;
Hoeijmakers, J. H. J.; Hanaoka, F. EMBO J. 1994, 13, 1831–1843.
(53) Volker, M.; Mone, M. J.; Karmakar, P.; van Hoffen, A.; Schul,
W.; Vermeulen, W.; Hoeijmakers, J. H.; van Driel, R.; van Zeeland, A. A.;
Mullenders, L. H. Mol. Cell 2001, 8, 213–224.
(54) Hey, T.; Lipps, G.; Sugasawa, K.; Iwai, S.; Hanaoka, F.; Krauss,
G. Biochemistry 2002, 41, 6583–6587.
(19) Dizdaroglu, M.; Jaruga, P.; Rodriguez, H. Free Radical Biol. Med.
2001, 30, 774–784.
(55) Riedl, T.; Hanaoka, F.; Egly, J. M. EMBO J. 2003, 22, 5293–
5303.
(20) Jaruga, P.; Birincioglu, M.; Rodriguez, H.; Dizdaroglu, M.
Biochemistry 2002, 41, 3703–3711.
(56) Yang, Z. G.; Liu, Y.; Mao, L. Y.; Zhang, J. T.; Zou, Y.
Biochemistry 2002, 41, 13012–13020.
(21) Kirkali, G.; de Souza-Pinto, N. C.; Jaruga, P.; Bohr, V. A.;
Dizdaroglu, M. DNA Repair 2009, 8, 274–278.
(22) Rodriguez, H.; Jaruga, P.; Leber, D.; Nyaga, S. G.; Evans, M. K.;
Dizdaroglu, M. Biochemistry 2007, 46, 2488–2496.
(23) Jaruga, P.; Xiao, Y.; Nelson, B. C.; Dizdaroglu, M. Biochem.
Biophys. Res. Commun. 2009, 386, 656–660.
(24) Brooks, P. J. DNA Repair 2008, 7, 1168–1179.
(25) Kirkali, G.; Tunca, M.; Genc, S.; Jaruga, P.; Dizdaroglu, M. Free
Radical Biol. Med. 2008, 44, 386–393.
(57) Yang, Z.; Roginskaya, M.; Colis, L. C.; Basu, A. K.; Shell, S. M.;
Liu, Y.; Musich, P. R.; Harris, C. M.; Harris, T. M.; Zou, Y. Biochemistry
2006, 45, 15921–15930.
(58) Gunz, D.; Hess, M. T.; Naegeli, H. J. Biol. Chem. 1996, 271,
25089–25098.
(59) Buterin, T.; Hess, M. T.; Luneva, N.; Geacintov, N. E.; Amin, S.;
Kroth, H.; Seidel, A.; Naegeli, H. Cancer Res. 2000, 60, 1849–1856.
(60) Dip, R.; Camenisch, U.; Naegeli, H. DNA Repair 2004, 3,
1409–1423.
(26) Jasti, V. P.; Das, R. S.; Hilton, B. A.; Weerasooriya, S.; Zou, Y.;
Basu, A. K. Biochemistry 2011, 50, 3862–3865.
(27) Gasparutto, D.; Bourdat, A. G.; D’Ham, C.; Duarte, V.; Romieu,
A.; Cadet, J. Biochimie 2000, 82, 19–24.
(28) Miaskiewicz, K.; Miller, J. H.; Fuciarelli, A. F. Nucleic Acids Res.
1995, 23, 515–521.
(29) Karwowski, B. T. THEOCHEM 2009, 915, 73–78.
(30) Karwowski, B. T. Org. Biomol. Chem. 2010, 8, 1603–1609.
(31) Birnbaum, G. I.; Cygler, M.; Dudycz, L.; Stolarski, R.; Shugar,
D. Biochemistry 1981, 20, 3294–3301.
(61) Buterin, T.; Meyer, C.; Giese, B.; Naegeli, H. Chem. Biol. 2005,
12, 913–922.
(62) Min, J. H.; Pavletich, N. P. Nature 2007, 449, 570–575.
(63) Brown, K. L.; Roginskaya, M.; Zou, Y.; Altamirano, A.; Basu,
A. K.; Stone, M. P. Nucleic Acids Res. 2010, 38, 428–440.
(64) Marion, D.; Wuthrich, K. Biochem. Biophys. Res. Commun. 1983,
113, 967–974.
(65) TOPSPIN, 3.1 ed.; Bruker Biospin, Inc.: Billerica, MA, 2010.
(66) Goddard, T. D.; Kneller, D. G. SPARKY; University of Cali-
fornia, San Francisco: 2006.
(32) Haromy, T. P.; Raleigh, J.; Sundaralingam, M. Biochemistry
1980, 19, 1718–1722.
(67) Piotto, M.; Saudek, V.; Sklenar, V. J. Biomol. NMR 1992, 2,
661–665.
(33) Karwowski, B. T. Tetrahedron: Asymmetry 2008, 19, 2390–
2395.
(34) Romieu, A.; Gasparutto, D.; Cadet, J. Chem. Res. Toxicol. 1999,
12, 412–421.
(68) Gorenstein, D. G. Methods Enzymol. 1992, 211, 254–286.
(69) Markley, J. L.; Bax, A.; Arata, Y.; Hilbers, C. W.; Kaptein, R.;
Sykes, B. D.; Wright, P. E.; Wuthrich, K. J. Biomol. NMR 1998, 12,
1–23.
(35) Reid, B. R. Q. Rev. Biophys. 1987, 20, 2–28.
(36) Patel, D. J.; Shapiro, L.; Hare, D. Q. Rev. Biophys. 1987,
20, 35–112.
(70) Keepers, J. W.; James, T. L. J. Magn. Reson. 1984, 57, 404–426.
(71) Liu, H.; Spielmann, H. P.; Ulyanov, N. B.; Wemmer, D. E.;
James, T. L. J. Biomol. NMR 1995, 6, 390–402.
(37) Boelens, R.; Scheek, R. M.; Dijkstra, K.; Kaptein, R. J. Magn.
Reson. 1985, 62, 378–386.
(72) Salazar, M.; Fedoroff, O. Y.; Miller, J. M.; Ribeiro, N. S.; Reid,
B. R. Biochemistry 1993, 32, 4207–4215.
(38) Griesinger, C.; Sorensen, O. W.; Ernst, R. R. J. Am. Chem. Soc.
1985, 107, 6394–6396.
(73) Wang, H.; Zuiderweg, E. R. P.; Glick, G. D. J. Am. Chem. Soc.
1995, 117, 2981–2991.
(39) Borgias, B. A.; James, T. L. J. Magn. Reson. 1990, 87, 475–487.
(40) Borgias, B. A.; James, T. L. Methods Enzymol. 1989, 176,
169–183.
(74) Geen, H.; Freeman, R. J. Magn. Reson. 1991, 93, 93–141.
(75) Lankhorst, P. P.; Haasnoot, A. G.; Erkelens, C.; Altona, C.
Nucleic Acids Res. 1984, 12, 5419–5428.
20367
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368

Journal of the American Chemical Society
ARTICLE
(76) Kirkpatrick, S.; Gelatt, C. D., Jr.; Vecchi, M. P. Science 1983,
220, 671–680.
(77) Frisch, M. J.; et al. GAUSSIAN; Gaussian, Inc.: Wallingford,
CT, 2004.
(78) Case, D. A.; Cheatham, T. E., 3rd; Darden, T.; Gohlke, H.; Luo,
R.; Merz, K. M., Jr.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J.
J. Comput. Chem. 2005, 26, 1668–1688.
(79) Bashford, D.; Case, D. A. Annu. Rev. Phys. Chem. 2000, 51,
129–152.
(80) Tsui, V.; Case, D. A. Biopolymers 2000, 56, 275–291.
(81) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey,
R. W.; Klein, M. L. J. Chem. Phys. 1983, 79, 926–935.
(82) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys.
1977, 23, 327–341.
(83) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 12,
10089–10092.
(84) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.;
Pedersen, L. G. J. Chem. Phys. 1995, 19, 8577–8593.
(85) Lavery, R.; Sklenar, H. J. Biomol. Struct. Dyn. 1988, 6, 63–91.
(86) Lu, X. J.; Olson, W. K. Nucleic Acids Res. 2003, 31, 5108–5121.
20368
dx.doi.org/10.1021/ja207407n |J. Am. Chem. Soc. 2011, 133, 20357–20368