Resistance of bulky DNA lesions to nucleotide
excision repair can result from extensive aromatic
lesion–base stacking interactions
Dara A. Reeves1, Hong Mu2, Konstantin Kropachev1, Yuqin Cai2, Shuang Ding2,
Alexander Kolbanovskiy1, Marina Kolbanovskiy1, Ying Chen1, Jacek Krzeminski3,
Shantu Amin3, Dinshaw J. Patel4, Suse Broyde2,* and Nicholas E. Geacintov1,*
1Department of Chemistry,2Department of Biology, New York University, 100 Washington Square East,
New York, NY 10003,3Department of Pharmacology, Pennsylvania State University, 500 University Drive,
Hershey, PA 17033 and4Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York,
NY 10065, USA
Received May 5, 2011; Revised June 7, 2011; Accepted June 9, 2011
The molecular basis of resistance to nucleotide
excision repair (NER) of certain bulky DNA lesions
is poorly understood. To address this issue, we have
studied NER in human HeLa cell extracts of two
topologically distinct lesions, one derived from
benzo[a]pyrene (10R-(+)-cis-anti-B[a]P-N2-dG), and
one from the food mutagen 2-amino-1-methyl-
embedded in either full or ‘deletion’ duplexes (the
partner nucleotide opposite the lesion is missing).
All lesions adopt base-displaced intercalated con-
formations. Both full duplexes are thermodynamic-
ally destabilized and are excellent substrates of
NER. However, the identical 10R-(+)-cis-anti-B[a]P-
N2-dG adduct in the deletion duplex dramatically
enhances the thermal stability of this duplex, and
is completely resistant
dynamics simulations show that B[a]P lesion-
induced distortion/destabilization is compensated
by stabilizing aromatic ring system–base stacking
interactions. In the C8-dG-PhIP-deletion duplex,
the smaller size of the aromatic ring system and
the mobile phenyl ring are less stabilizing and yield
moderate NER efficiency. Thus, a partner nucleotide
opposite the lesion is not an absolute requirement
for the successful initiation of NER. Our observa-
tions are consistent with the hypothesis that car-
contribute to the local DNA stability, can prevent
to NER. Molecular
the successful insertion of an XPC b-hairpin into
the duplex and the normal recruitment of other
downstream NER factors.
Bulky DNA lesions are subject to removal by the nucleo-
tide excision repair (NER) machinery in order to maintain
the integrity of the genome. There are two NER pathways,
transcription-coupled repair (TCR) and global genomic
repair (GGR). In TCR (1,2), the lesions cause the RNA
polymerase to stall, which results in the recruitment of
other NER factors that remove the lesions. In GGR (3),
the local distortion/destabilization of the DNA caused by
the lesions is recognized by the NER factors XPC/Rad23B
(5); these factors bind to the site of the lesion, and cause a
local strand separation involving ?6bp in the case
of benzo[a]pyrene (B[a]P)-derived adducts (4). Other
factors are subsequently recruited to this complex and
ultimately produce dual incisions and the excision of
lesion-containing oligonucleotides that are 24–32nt in
The structural properties of bulky DNA lesions that
elicit NER are the subject of considerable interest, particu-
larly since the relative incision efficiencies for structurally
different adducts vary over several orders of magnitude
(3,7,8). Moreover, the base sequence context in which a
given lesion is embedded can modulate the repair
efficiencies (9,10). A recently determined crystal structure
of a truncated form of yeast Saccharomyces cerevisiae
Rad4/Rad23 (11), an ortholog of the mammalian XPC/
Rad23B, in a complex with an oligonucleotide containing
a T<>T cyclobutane pyrimidine dimer (CPD) lesion,
*To whom correspondence should be addressed. Tel: +1 212 998 8407; Fax: +1 212 998 8421; Email: email@example.com
Correspondence may also be addressed to Suse Broyde. Tel: +1 212 998 8231; Fax: +1 212 995 4015; Email: firstname.lastname@example.org
Nucleic Acids Research, 2011, Vol. 39, No. 20Published online 15 July 2011
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
reveals that a b-hairpin is inserted into the DNA helix that
stabilizes the separation between the damaged and com-
plementary undamaged strand. The position of the lesion
could not be determined, but the two mismatched thymine
bases in the complementary strand opposite the CPD were
flipped out of the duplex and in contact with amino acid
residues in two of the three b-hairpins in this complex. In
prokaryotic NER, a crystal structure of a complex of the
Bacillus caldotenax UvrB bound to double-stranded
damaged DNA (12) also shows b-hairpin intrusion
between the two strands and partner base flipping as an
element in lesion recognition. It has therefore been sug-
gested that the flipped out bases in the complementary
strand that are normally positioned opposite the lesion
may play a role in the recognition step of both prokaryotic
and eukaryotic NER (11–14). Indeed, base-displaced
intercalated 10S-(?) and 10R-(+)-cis-anti-B[a]P-N2-dG
double-stranded DNA, in which both the damaged base
and the partner base are extruded from the double helix,
are excellent NER substrates (13,15). However, the same
cis-B[a]P-dG adducts are completely resistant to NER in
human cell extracts when the nucleotide dCp opposite the
lesion is removed (13,15). Such deletion duplexes might be
biologically significant since they could arise during DNA
replication in vivo when DNA polymerases fail to insert a
dNTP opposite bulky lesions, and skip instead to the next
50-downstream unmodified template base (16–18).
In this work we explored the molecular basis of the re-
sistance of bulky lesions to NER and the role of a
flipped-out base, by studying the repair efficiencies of a
DNA adduct derived from the heterocyclic aromatic
(PhIP), a member of a different class of bulky carcinogenic
(PAHs). Such heterocyclic aromatic amines (HAAs) are
highly potent mutagens and suspected human carcino-
gens, and are present in cigarette smoke and in meats
Figure 1. Structures and sequences of cis-B[a]P-dG and C8-dG-PhIP-modified duplexes. (A) Chemical structure of the cis-B[a]P-dG adduct. The
glycosidic torsion angle, ? for both modified duplexes is defined as O40?C10?N9?C4. The B[a]P-dG linkage site torsion angles, a0and b0, are defined
as follows: a0=N1(G)?C2(G)?N2(G)?C10(B[a]P) and b0=C2(G)?N2(G)?C10(B[a]P)?C9(B[a]P). (B) Chemical structure of the C8-dG-PhIP
adduct.ThePhIP-dG linkage sitetorsion angles,
b0=C8(G)?N(PhIP)?C2(PhIP)?N1(PhIP). The torsion angle g0is defined as C7(PhIP)?C6(PhIP)?C10(PhIP)?C20(PhIP). (C) Sequence I.
Sequence context of the 11/11-mer full duplex. (D) Sequence II. Sequence context of the 11/10-mer deletion duplex. G6*, colored in red, represents
the lesion-modified guanine. (E) NMR solution structures for the cis-B[a]P-dG-modified 11/11mer full and 11/10mer deletion duplexes (34,35) and
C8-dG-PhIP 11/11mer full duplex (33) utilized as initial structures for the MD simulations. The C8-dG-PhIP deletion duplex was modeled based on
the cis-B[a]P-dG deletion duplex NMR structure (35), as described in ‘Materials and Methods’ section. The duplexes are viewed from the minor
groove side. Hydrogen atoms and phosphate oxygen atoms are deleted for clarity. In the modified full duplexes, the lesion strand backbone is colored
in light gray and the complementary strand is colored in dark gray. Phosphate atoms are colored purple. The adduct is colored red, the modified
guanine and its partner base are colored cyan, and the neighboring bases are colored blue.
Nucleic Acids Research, 2011,Vol.39, No. 20 8753
and fish cooked at high temperatures (19–24). Following
metabolic activation (25–30), PhIP forms several covalent
adducts with DNA (31,32), of which the C8-dG-PhIP
adduct (Figure 1B) is the most prevalent and only well
characterized lesion (31,32). An NMR solution structure
of the C8-dG-PhIP adduct (G*) has revealed that ?90%
of the population of this adduct in sequence I (Figure 1C)
adopts a base-displaced intercalated conformation (33),
which is in the same family of adduct conformations as
the cis-B[a]P-dG adduct (34,35) (Figure 1A). However it is
noteworthy that the topology of the C8-dG-PhIP adduct
with its fewer aromatic rings, elongated ring system, bulky
methyl group, and mobile phenyl ring (Figure 1B) is very
different from that of the cis-B[a]P-dG adduct (Figure 1A).
We compare here the NER of the C8-dG-PhIP and the
cis-B[a]P-dG adducts in full and deletion duplexes in the
sequences defined in Figure 1C and D. We show that both
of these lesions, positioned in fully complementary
duplexes of the identical sequence context, are excised
with similar high efficiencies in human NER in HeLa
cell extracts. While the cis-B[a]P-dG adduct is completely
resistant to NER in the deletion duplex (Figure 1D), the
PhIP adduct positioned in the identical deletion duplex is
excised, but with an efficiency that is reduced to 15–20%
of that in the full duplex. In order to examine the origins
of the differences in the NER response, we determined the
impact of these lesions on the thermal stabilities of the
double-stranded DNA. We find that both lesions destabil-
ize the full duplexes to near-similar extents; however, the
two lesions stabilize the deletion duplexes to significantly
different extents, with the cis-B[a]P-dG adduct being more
stabilizing. To gain molecular insights on the structural
and dynamic properties of the lesions that govern their
modeling and molecular dynamics (MD) simulations for
the full and deletion duplexes (Figure 1E). These simula-
tions pinpoint differences in structural distortions and
helix-stabilizing/destabilizing properties, and thereby de-
lineate the characteristics of the lesions that elicit resist-
ance to NER.
MATERIALS AND METHODS
Synthesis of modified oligonucleotide sequences and
preparation of NER-substrates
The synthesis of the site-specifically modified 11-mer 50-C
CATCGCTACC to generate the centrally positioned
C8-dG-PhIP adduct was accomplished by the methods
described by Brown et al. (31). Briefly, N-acetoxy-PhIP
was dissolved in methanol (1mg/ml) and added gradually
(aliquots added over a 5h period) to a solution containing
the 11-mer oligonucleotide, in 10mM sodium citrate
buffer solution containing 1mM EDTA and 0.1M NaCl
at pH 7.0 for 60min at 37?C. The desired modified oligo-
nucleotides were separated and purified by reversed phase
HPLC methods utilizing a Microsorb-MV 100-5 C18
C8-dG-PhIP adducts embedded in the 11-mer oligo-
adducts in the same 11-mer oligonucleotides were
prepared as described in detail elsewhere (36). For the
thermal melting experiments,
carcinogen-modified 11-mers were annealed with their
fully complementary 11-mer strand 50-GGTAGCGAT
GG, or the 10-mer strand 50-GGTAGGATGG to
generate the full and deletion duplexes, respectively. The
135-mer full duplexes (the full sequence is depicted in
Supplementary Figure S5) used in the NER experiments
were prepared by annealing—ligation techniques, using
two 67-mer oligonucleotides and the centrally positioned,
modified and32P-labeled 11-mer 50-32p-CCATCG*CTAC
C (where G* denotes the site of the lesion) and a fully
complementary 135-mer duplex as described in detail by
Kropachev et al. (37). The analogous deletion duplexes
were generated in the same manner, except that the dCp
nucleotides in the complementary strands opposite the
lesion were missing. In all cases, the adducts were pos-
itioned at the 67th nucleotide counted from the 50-side.
Thermal stabilities of oligonucleotide duplexes
Melting curves of the modified duplexes were measured
using UV absorbance methods based on Kolbanovskiy
et al. (38). Briefly, the unmodified 50-CCATCGCTACC
annealed to the complementary strand 50-GGTAGCGA
TGG-30in a 1:1 ratio of modified:complementary strand
DNA, in 20mM phosphate buffer (pH 7.0) containing
100mM NaCl. Melting curves were generated using the
Cary 100 Bio UV/Vis spectrophotometer. The tempera-
ture was increased by 0.1?C increment per minute,
followed by 2s average reading times of the absorbance
at 260nm with the buffer solution being used as the
Human NER assays in HeLa cell extracts
These NER assays were conducted as described by
Kropachev et al. (37). Briefly, each reaction contained
1pmol of P32-internally labeled 135mer duplex with and/
or without adduct, in an aqueous mixture generated by
mixing 17.5ml of a 1M KCl solution with 20ml of a 10mM
Tris–ATP (pH 7.9) and 10ml freshly prepared cell extract
solution containing 40–45mg of protein), and sufficient
dialysis buffer (12mM MgCl2, 25mM HEPES–KOH pH
7.9, 2.5mM Dithiothreitol, 1mM EDTA 10% glycerol) to
produce a final volume of 50ml. After appropriate incuba-
tion times and subsequent preparation of the samples (37),
the reaction products were loaded onto a 12% polyacryl-
amide, 8M urea denaturing gel. The gels were dried,
exposed, and analyzed using a Storm 840 phosphoimager
and Image-Quant software.
Molecular modeling and MD simulations
C8-dG-PhIP and the cis-B[a]P-dG 11/11-mer full and
11/10-mer deletion duplexes, utilizing NMR solution
structures of these adducts as the basis for the initial
models (33–35) (Figure 1E). The C8-dG-PhIP deletion
duplex (Figure 1E) was modeled based on the NMR struc-
ture of the cis-B[a]P-dG deletion duplex (35), since no
performed25ns ofMDsimulations forthe
8754Nucleic Acids Research, 2011,Vol.39, No. 20
NMR structure was available. Full details of the molecu-
lar modeling, force fields and MD protocols, are given in
the Supplementary Data (Supplementary Tables S1 and
S2). For all cases, the MD simulations achieved good sta-
Structural ensembles from 10.0 to 25.0ns were employed
for analyses. Details of the analyses are provided in the
themean, after 10.0ns.
The C8-dG-PhIP and cis-B[a]P-dG adducts destabilize
the full duplexes but stabilize the deletion duplexes to
The impacts of these lesions on the duplex thermodynamic
properties are reflected in the melting points, Tm, of rela-
tively short oligonucleotide duplexes (39–41). In addition,
hyperchromicities (percentage of difference in the absorb-
ance measured at 260nm of single- and double-stranded
oligonucleotides) are indicative of the extent of base
stacking interactions in double-stranded DNA. Typical
C8-dG-PhIP or cis-B[a]P-dG adducts are depicted in
Figure 2A. The melting points of these duplexes specify
the temperature at which 50% of the single-stranded mol-
ecules are in the double-stranded and single-stranded
forms, and are summarized in Table 1. The changes in
the melting points relative to the unmodified duplexes
are defined by ?Tm=Tm(modified) – Tm(unmodified)
and are summarized in Figure 2B and Table 2. The
Tm-values of the C8-dG-PhIP and the cis-B[a]P-dG-
modified 11/11-mer full duplexes are lower by ?10?and
?12?, respectively, than those of the unmodified duplexes.
Thus, both lesions destabilize the full duplexes to almost
the same extent. The hyperchromicity values of the
(12%) and unmodified (14%) full duplexes measured at
260nm are similar (Table 1), indicating that the overall
impact of both lesions on the DNA base stacking inter-
actions in these 11/11-mer full duplexes is not large, except
at the site of the lesion.
In contrast to the destabilizing impact of the lesions on
the thermodynamic stabilities that are reflected in the Tm-
values of the modified 11/11-mer full duplexes, the
modified 11/10-mer deletion duplexes are stabilized by
the same lesions (Figure 2). While the Tmof the unmodi-
fied deletion duplex is 24?C lower than that of the unmodi-
fied 11/11-mer full duplex (Table 1), both kinds of lesions
stabilize the deletion duplexes. The C8-dG-PhIP lesion
stabilizes the deletion duplex modestly by ?Tm? +3?C
(Figure 2B and Table 2), and the cis-B[a]P-dG lesion more
prominently by ?Tm? +19?C (Figure 2B and Table 2).
deletion duplex (11%) is 3% points lower than that of
the full duplex (Table 1), as expected from the loss of
base-base stacking interactions in the deletion duplex.
Remarkably, the hyperchromicity rises to 14% in the
cis-B[a]P-dG deletion duplex (Table 1), which is the
same as the value for the full unmodified duplex; this
suggests an increase in stacking due to interactions
between the B[a]P residue and the neighboring bases,
which dramatically stabilize the cis-B[a]P-dG deletion
duplex. The enhanced stability of the deletion duplex is
indicated by the prominent increase in the Tm-value (Table
1). Overall, these results clearly point out that bulky poly-
cyclic aromatic DNA lesions exert not only destabilizing
effects by distorting the normal B-DNA helical structure,
but are also capable of stabilizing the same duplexes via
intermolecular van der Waals (vdW) stacking and other
interactions with DNA residues.
Figure 2. Thermal
C8-dG-PhIP-modified duplexes. (A) UV melting profiles of unmodified
(Unmod), cis-B[a]P-dG (B[a]P) and C8-dG-PhIP (PhIP) modified
deletion duplexes measured at 260nm. The full (Full) and deletion
(Del) duplexes are defined in Figure 1 and the Tm values and
hyperchromicity values are summarized in Table 1. (B) Comparisons
of the ?Tm=Tm (modified) – Tm (unmodified values) based on
thermal melting data of Table 1. The melting points of the duplexes,
Tm, are averages of 2–3 experiments.
meltingpropertiesof cis-B[a]P-dG and
Table 1. Tmand hyperchromicity values of unmodified, C8-dG-PhIP
and cis-B[a]P-dG-modified full (Full) and deletion duplexes (Del)
Nucleic Acids Research, 2011,Vol.39, No. 208755
Full duplexes are excellent substrates of NER while in
deletion duplexes the B[a]P adduct is fully resistant and
the PhIP adduct elicits only moderate repair
The incubation of 135-mer duplexes (Supplementary
cis-B[a]P-dG lesions in HeLa cell extracts gives rise to
the appearance of radioactively labeled oligonucleotide
dual incision fragments 24–32nt in length that can be
resolved from the 135-mer duplexes by denaturing gel elec-
trophoresis (Figures 3A and B). A small fraction (<10%)
of the C8-dG-PhIP adducts, but not the cis-B[a]P-dG
adducts, decompose during the workup of the samples
subsequent to the incubation reaction. This is evident
from the appearance of 67-mer oligonucleotide cleavage
fragments in denaturing gels that appear even in the case
of untreated control samples (Figure 3A). Furthermore,
the fractions of these decomposition products are inde-
pendent of incubation time in NER-viable cell extracts,
while the fractions of NER incision products increase
linearly with incubation time in the same extracts
(Supplementary Figures S6 and S7).
Examples of typical densitometry tracings of the 30min
gel electrophoresis lanes are depicted in Figure 3B.
Comparisons of the relative extents of NER were
calculated from the integration of the radioactivity in
divided by the total radioactivity in each lane of the gel
autoradiogram (Figure 3A). The NER dual incision
efficiencies varied from 0% to ?5–8% in the experiments
reported here. In order to achieve reproducibility and to
take account of varying activities of different cell extracts,
the NER efficiencies in each gel autoradiogram were
normalizedto the value
C8-dG-PhIP-modified 135/135-mer full duplexes after an
incubation time of 30min. These values were arbitrarily
assigned a value of 100, and all other NER efficiencies are
reported relative to this value for each gel. The relative
efficiencies of dual incisions of the C8-dG-PhIP and
cis-B[a]P-dG-modified 135/135-mer full duplexes increase
as a function of incubation time within the range of
0–30min (Figure4). The
B[a]P-dG-modified 135/134-mer deletion duplex to NER
is evident from the flat densitometry trace shown in
Figure 3B, which is indistinguishable from the base line.
Thus, NER is completely abolished for this adduct
(13,15). In contrast, the C8-dG-PhIP-modified deletion
duplex is incised in the cell extracts (Figures 3A and 4).
However, at all incubation times the incision efficiencies
are uniformly 5–6 times lower in the C8-dG-PhIP-
PhIP-modified full duplexes (Figure 4).
single C8-dG-PhIP and
Molecular modeling and MD simulations
In order to obtain structural, dynamic and energetic
understanding of the experimental
and NER studies, we performed molecular modeling and
cis-B[a]P-dG adducts, in 11/11-mer full and 11/10-mer
deletion duplexes. These were based on their base-
displaced intercalated solution structures (Figure 1E)
that were determined by NMR methods (33–35). There
is no experimental NMR data for the C8-dG-PhIP
deletion duplex. As detailed in the Supplementary Data,
the known cis-B[a]P-dG deletion duplex NMR solution
structure (35) was utilized to obtain an initial structure
for MD simulation of the C8-dG-PhIP deletion duplex,
byreplacing the B[a]P-modified
PhIP-modified guanine (Figure 1E). We carried out
detailed analyses of the MD trajectories between 10 and
25ns during which the simulations were stable, as shown
by the RMSD data for each 25ns MD simulation
(Supplementary Figure S8).
Base-displaced intercalated conformations affect duplex
structures and stabilities differently depending on adduct
topology and type of duplex
MD simulations show base-displaced intercalated con-
formations of the C8-dG-PhIP and cis-B[a]P-dG adducts
in full and deletion duplexes (Figures 5 and 6). These
simulations indicate that there are structural features
B[a]P-derived duplexes although both are intercalated
with the displacement of the damaged bases and their
partners fromtheir normal
The topological differences between the lesions are
notable: the PhIP is more elongated, contains only three
aromatic rings, one of which is the mobile phenyl ring,
while the B[a]P is rigid and planar, and contains four
fully aromatic rings. While the overall features of the
C8-dG-PhIP and cis-B[a]P-dG-modified full and deletion
duplexes shown in the ensembles of structures derived
from the MD simulations are similar to those of their
respective NMR solution structures (33–35), the simula-
tions provide important new insights into the lesion
ring. In the full duplex, the PhIP phenyl ring protrudes
into the minor groove towards the complementary strand
and rotates rapidly. This rapid rotation is reflected by
the dynamic oscillation, between 38.4?and ?39.9?, of
the g0torsion angle (Figure 1B) shown in Figure 5A and
Movie S1 in Supplementary Data. Both G6* and its
partner base C17 are displaced into the major groove of
the full duplex. G6* stacks with base C5 on its 50-side,
while the inserted imidazopyridine (IP) ring of PhIP
stacks mainly with G18 in the helix. In the deletion
duplex, the phenyl ring also rotates rapidly, with the g0
torsion angle (Figure 1B) adopting values between 44.1?
and ?34.8?. The dynamics of this rotation is shown in
Figure 5B and Movie S2 in Supplementary Data.
Furthermore, the PhIP phenyl ring protrudes more into
the minor groove than in the full duplex, because the
absence of dC17 compresses the partner strand. In
concert, G6* protrudes further into the major groove
away from C5 and the PhIP IP ring now stacks with C5,
as well as with G16 (Figure 5B Top view). Modest
stacking interactions between the IP ring and other
adjacent bases are equivalent in the full and the deletion
8756Nucleic Acids Research, 2011,Vol.39, No. 20
stacking interactions involving C5 and G16 provide
stabilization to the deletion duplex containing this
adduct, as revealed in the thermal melting data (Figure 2
and Table 1).
cis-B[a]P-dG-modified duplexes: planar, rigid and bulky
B[a]P ring. The cis-B[a]P-dG adduct contains more
aromatic rings, is less elongated than the PhIP residue,
andis planar andrigid
B[a]P-dG-modified full duplex, the G6* base is displaced
into the minor groove, while C17 is displaced into the
major groove; however,
pronounced than in the case of the C8-dG-PhIP-
modified full duplex (Figures 5A and 6A). In the
backbone, G16 and G17 collapse onto each other and
(Figure 6B). The B[a]P aromatic ring system is shifted
more towards the complementary strand and more into
the minor groove than in the full duplex (Figure 6A
and B). These conformational changes provide better
aromatic ring overlap between B[a]P rings and neighbor-
ing bases, primarily G17 and the periphery of G16, in the
deletion duplex than in the full duplex (Figure 6A and B
and Supplementary Table S3); this results in stabilization
that is manifested in the thermal melting points (Figure 2
and Table 1).
(Figure 1).Inthe cis-
this displacementis less
around theB[a]P rings
Stacking interaction energies between the aromatic ring
systems of the lesions and adjacent base pairs differ in
full and deletion duplexes
In order to gain deeper insights into the effects of PAH
adduct ring system topology on stacking interactions
Figure 3. NER assays in Human HeLa cell extracts. (A) Autoradiogram of a representative denaturing 12% polyacrylamide gel showing incisions of
full (Full) and deletion (Del) 135-mer duplexes containing single cis-B[a]P-dG (B[a]P) and C8-dG-PhIP (PhIP) adducts in HeLa cell extracts at 10, 20
and 30min of incubation. The zero time point samples serve as controls (these samples were incubated in heat-deactivated cell extracts, but otherwise
underwent the same treatment). Lane Marker: oligonucleotide markers of different lengths. (B) Densitometry tracings of the 30min lanes shown in
the autoradiogram in Figure 3A. M: markers.
Figure 4. Incision kinetics of the internally labeled 135-mer modified
duplexes in HeLa cell extracts. The incision efficiencies were normalized
in each of the four independent experiments to the value obtained with
the C8-dG-PhIP full duplex (relative value of 100) at the 30min
incubation time point. The averages and standard deviations shown
were obtained from these normalized values. Note that the cis-
B[a]P-dG deletion duplex is not incised.
Nucleic Acids Research, 2011,Vol.39, No. 208757
between the adduct and the adjacent base pairs, we
computed the vdW interaction energy (EvdW) between
the adduct aromatic rings and all the adjacent bases; this
energy was computed for each of the distinct structures
(total of 15000) that evolve during the MD simulations
from 10 to 25ns, as detailed in Supplementary Data.
Figure 7A shows the number of structures from the total
ensemble at each given vdW interaction energy value, and
is called a population distribution. As expected, vdW
interaction energies are weaker for the elongated PhIP
with its dynamically mobile phenyl ring than for the
rigid, planar and more aromatic B[a]P ring system. This
dynamic, non-planar ring protrudes into the minor
groove, and forces the PhIP IP rings toward this groove;
consequently, stacking interactions between the PhIP ring
system and neighboring bases is diminished. It is evident
that the vdW interaction energies between the lesion
aromatic rings and the adjacent bases are stronger in the
deletion duplexes than the full duplexes in each case. This
stems from the compressed backbone of the partner strand
in the deletion duplex, which allows better aromatic ring
system overlap between the lesion and adjacent bases in
both cases, as detailed above. The lack of the dCp nucleo-
tide opposite the lesion allows for better B[a]P – base
stacking interactions, thus pointing to the destabilizing
influence of the partner dCp nucleotide opposite the
lesion in the full duplex. The contributions to the
stacking energyof each
Supplementary Table S3.
Watson–Crick base pairing is impacted differently
depending on the lesion and type of duplex
bonding is an important NER recognition signal (37,42).
Therefore, for both PhIP and B[a]P adducts, in full and
deletion duplexes, we have evaluated the impact of the
lesion on the local hydrogen bond quality for the base
pairs flanking the site of the lesion, namely C5:G18 and
C7:G16 in full duplexes and C5:G17 and C7:G16 in
distortion ofnormal Watson–Crick hydrogen
Figure 5. Molecular dynamics of the C8-dG-PhIP-modified full and deletion duplexes. Hydrogen atoms and phosphate oxygen atoms are deleted for
clarity. The lesion strand backbone is in light gray and the complementary strand is in dark gray. Phosphate atoms are purple. The adduct is red, the
modified guanine and its partner base are cyan, and the neighboring bases are blue. (A) Dynamics of phenyl ring of the C8-dG-PhIP-modified full
duplex. The structures with the highest and lowest g0values (38.4?and ?39.9?) are shown on the left to emphasize the dynamics of the phenyl ring.
The central trimer is viewed from the minor groove side (left) and along the helix axis (Top view, right). The time dependence of the g0torsion angle
is shown on the right. (B) Dynamics of phenyl ring of the C8-dG-PhIP-modified deletion duplex. The structures with the highest and the lowest g0
values (44.1?and ?34.8?) are shown on the left to emphasize the dynamics of the phenyl ring. The central trimer is viewed from the minor groove
side (left) and along the helix axis (Top view, right). The time dependence of the g0torsion angle is shown on the right.
8758 Nucleic Acids Research, 2011,Vol.39, No. 20
deletion duplexes. We utilized the hydrogen bond quality
index (IH) developed by us previously (43), to evaluate the
deviation of the hydrogen bonds for a selected base pair
from ideal Watson–Crick
computed the IHsum over the 15000 structures collected
between10 and 25ns
Supplementary Data). Ideal Watson–Crick hydrogen
bonds have an IH-value of zero. The more the hydrogen
bond distances and angles differ from those in ideal
Watson–Crick hydrogen bonds, the larger the value of
IH. Such deviations in distances and angles are produced
by various combinations of disturbances to the global
base–base parameters, such as shear, stretch, stagger,
buckle, propeller and opening, which can vary in concert
(44) (Supplementary Figure S9).
hydrogen bonding; we
of MD simulation (see
The mobility of the PhIP phenyl ring disturbs the base pairs
flanking the lesion. In the C8-dG-PhIP-modified full
duplex, distortion of the C7:G16 base pair is indicated
by a higher trajectory summed IH-value than that of the
analogous C7:G16 base pair in the unmodified duplex
(Figure 7B). The origin of these distortions is the rotation-
al mobility of the phenyl ring (Figure 5A): this mobility
causes episodic crowding between the hydrogen atoms of
the phenyl ring and G16, causing disturbances to the
C7:G16 base pair, especially in buckle and propeller
twist (Supplementary Figure S9 and Movie S1 in
Supplementary Data). The methyl group also contributes
to the distortion of the C7:G16 hydrogen bonds through
close interaction with the sugar ring of dC7, thus
somewhat displacing this base from its normal position
relative to its partner G16. However, the other flanking
(Figure 5A), with an IHvalue comparable to that of the
unmodified duplex (Figure 7B) since both the phenyl and
methyl groups are further away from this base pair. In the
C8-dG-PhIP-modified deletion duplex, the IHvalues of
the C5:G17 and C7:G16 base pairs are modestly higher
than for the C5:G18 and C7:G16 base pairs in the un-
modified duplex (Figure 7B); this suggests small distor-
tions of Watson–Crick hydrogen bonding caused by the
rapid rotation of the phenyl ring in the minor groove of
the deletion duplex, which produces episodic crowding
between hydrogen atoms of the phenyl ring and the
guaninebases G16 and
Supplementary Data). However, the crowding is less
severe than in the full duplex, because the phenyl ring
protrudes more into the minor groove in the deletion
duplex and therefore does not significantly perturb the
normal position of G16. Due to the compression of the
complementary strand, G17 partially collapses onto G16.
Base pairs flanking the cis-B[a]P-dG adduct are disturbed
in the full duplex but scarcely in the deletion duplex. In the
cis-B[a]P-dG-modified full duplex, the IH values of the
C5:G18 and C7:G16 base pairs flanking the adduct
Figure 6. Representative structures of the cis-B[a]P-dG-modified full and deletion duplexes. Hydrogen atoms and phosphate oxygen atoms are
deleted for clarity. The lesion strand backbone is in light gray and the complementary strand is in dark gray. Phosphate atoms are purple. The
adduct is red, the modified guanine and its partner base are cyan, and the neighboring bases are blue. (A) Best representative structure of the
cis-B[a]P-dG-modified full duplex viewed from the minor groove and along the helix axis. The best representative structure viewed along the helix
axis (Top view) is shown on the right to emphasize stacking interactions with neighboring bases. (B) Best representative structure of the
cis-B[a]P-dG-modified deletion duplex viewed from the minor groove, along the helix axis and from the major groove. The best representative
structure viewed along the helix axis (Top view) is shown in the middle to emphasize stacking interactions with neighboring bases. The best
representative structure viewed from the major groove side is shown on the right to emphasize the wedge-like shape induced by the deletion.
Nucleic Acids Research, 2011,Vol.39, No. 208759
are the highest observed in our simulations and thus are
the most perturbed (Figure 7B). The origin of the disturb-
ance to the C5:G18 base pair is the steric crowding
between G18 and the hydrogen atom at the N2-G6*
linkage site, as well as the N1 hydrogen atom of G6*
(Movie S3 in Supplementary Data). These close contacts
cause the C5:G18 base pair to episodically buckle and
propeller twist. The C7:G16 base pair also buckles and
Figure 7. Stabilizing and destabilizing properties of lesions in modified duplexes. (A) The population distribution of the vdW interaction energy
between adduct aromatic rings and the neighboring bases. The vdW interaction energies were calculated as described in Supplementary Data.
Energies for the cis-B[a]P-dG (B[a]P) and C8-dG-PhIP (PhIP) modified full duplexes (Full) are represented in dark green and purple, and in light
green and magenta for the respective deletion duplexes (Del). Mean values and standard deviations (kcal/mol): B[a]P Del, ?27.9±2.6; B[a]P Full,
?25.5±2.6; PhIP Del, ?22.4±2.1; PhIP Full, ?19.4±2.2. (B) Trajectory summed hydrogen bond quality index for the C:G base pairs 50and 30to
the lesion in the cis-B[a]P-dG (B[a]P) and C8-dG-PhIP (PhIP) modified deletion (Del) and full (Full) duplexes, and for the C:G base pairs at
comparable positions in the unmodified duplex. The trajectory summed hydrogen bond quality indexes were calculated as described in the
Supplementary Data. The specific base pairs are labeled at the top of the bars. The bars are color-coded as in Figure 7A.
Table 2. Summary of experimental NER efficiencies, ?Tmand structural/energetic properties of the C8-dG-PhIP and cis-B[a]P-dG adducts in
full (Full) and deletion (Del) duplexesa
Rigid, more aromatic rings
Mobile phenyl and methyl groups
Mobile phenyl and methyl groups
Rigid, more aromatic rings
aThe relative incision efficiencies are the same as shown in Figure 4 at 30min of incubation. ?Tmvalues are the same as shown in Figure 2B.
For each modified DNA duplex, ?IHis the trajectory summed IHvalues of both G:C base pairs flanking the lesion minus the trajectory summed IH
values of the G16:C7 and G18:C5 base pairs in the unmodified duplex, and was obtained as described in the Supplementary Data. Stacking
interaction energies are the mean values of the vdW interaction energies shown in Figure 7A.
8760Nucleic Acids Research, 2011,Vol.39, No. 20
propeller twists episodically because of steric crowding
between the amino group of G16 and the hydrogen
atom on the 7-hydroxyl group of the benzylic ring of the
B[a]P aromatic residue (Movie S3 in Supplementary
Data). By contrast, the hydrogen bond quality index IH
values of the C5:G17 and C7:G16 base pairs flanking the
aromatic B[a]P ring system in the cis-B[a]P-dG-modified
deletion duplex are not much different from the IHvalues
of the C5:G18 and C7:G16 base pairs in the unmodified
duplex: the C5:G17 base pair is slightly lower and the
C7:G16 base pair is slightly higher (Figure 7B). This dif-
ference arises from the backbone compression in the
deletion duplex, which forces the benzylic ring to
protrude further into the minor groove than in the full
duplex (Figure 6B); as a result, there is much less steric
crowding in the deletion duplex than in the full duplex
(Movie S4 in Supplementary Data). The collapse is
greater than for the C8-dG-PhIP deletion duplex,
described above, because the interaction of G16 and
G17 with the mobile phenyl ring prevents the greater
collapse seen in the cis-B[a]P-dG deletion duplex.
NER of base-displaced intercalated full and deletion
(dG-C8-AP) (46), 10S-(?) (47) and 10R-(+)-cis-anti-
intercalated conformations in full duplexes have been
shown to be well repaired in human HeLa cell extract
assays (13,15,48,49) or in the prokaryotic UvrABC
system (50). Although each adduct has its unique struc-
base-displaced intercalation appear to offer similar recog-
nition elements to the NER system. Several deletion
duplexes, which involve B[a]P-derived stereoisomeric
namely the 10S (+)-trans-anti-B[a]P-N2-dG and the 10S-
(?)-cis-anti-B[a]P-N2-dG ((?)-cis-B[a]P) adducts (15). The
intercalated conformation (51). While there is no NMR
solution structure available for the (?)-cis-B[a]P adduct in
the deletion duplex, its comparable repair inhibition and
thermal stabilization relative to the unmodified deletion
duplex (Table 1) (Tm=44±0.5?C, ?Tm=16.7?C, un-
published data) to the (+)-cis-B[a]P-dG deletion duplex
investigated here, indicate that their overall structural
characteristics are most likely similar. Additionally, the
NER efficiency in a dG-C8-AP deletion duplex with the
prokaryotic UvrABC system is quite inefficient compared
to the same adduct with a C opposite this lesion (50), and
the deletion duplex issignificantly
?4kcal/mol) according to thermal melting studies (52).
While the deletion duplexes share the feature of repair
resistance in human HeLa cell assays, our results here
show that unique topological elements can play an import-
ant part in determining susceptibility to NER.
bulky DNAadducts, includingN-
structural properties of
Recognition and verification steps in NER
The efficiencies of dual incisions catalyzed by the complex,
multi-step mammalian global genomic NER system is
often described in terms of a bipartite recognition mech-
anism (53,54). The first step involves the recognition of the
distorted/destabilized lesion site by XPC/Rad23B; the
second is believed to be the verification that a lesion
exists, by a mechanism involving helicases in the TFIIH
multi-protein complex (55). Following recognition by
B[a]P-dG adduct is opened within a 6bp stretch contain-
ing the lesion (4), and the adducted duplex is no longer
double-stranded. In the case of our cis-B[a]P-dG (or
C8-dG-PhIP) full and deletion duplexes, the verification
step is unlikely to contribute to the observed striking dif-
ferences in NER efficiencies, since the lesions are identical
in each pair of full and deletion duplexes but the NER
efficiencies are markedly different. However, the recruit-
ment of subsequent NER factors might be influenced by
the structural featuresof the initial
complexes formed, and these features may exert an
effect on the efficiencies of dual incisions (5).
duplex containinga cis-
Local thermodynamic stabilization may hinder b-hairpin
A common structural feature of both prokaryotic and eu-
karyotic NER mechanisms revealed in crystal structures is
the insertion of a b-hairpin of UvrB (12), or the yeast
Rad4/Rad23 (11) (an ortholog of the mammalian XPC/
Rad23B recognition factors) between the two strands of
the duplex at or close to the lesion site. The insertion of
the b-hairpin, with the concomitant extrusion of the bases
opposite the lesion in the complementary strand and their
interactions with the protein, stabilize the DNA–protein
complex. These structural properties suggest the hypoth-
esis that the local thermodynamic stability of the damaged
site within the modified DNA duplex determines the ease
or difficulty of DNA strand separation and concerted base
flipping (11,56); the insertion of the b-hairpin is facilitated
or hindered by the local thermodynamic stability. Our
results reveal that the severe distortions/destabilizations
produced by complete rupturing of Watson–Crick base
pairing, observed in our full duplexes, favor b-hairpin in-
sertion and thus the subsequent cascade of NER steps that
lead to the dual incisions of the damaged strand; this is
because stacking of the adduct aromatic ring system with
adjacent base pairs is insufficient to compensate energet-
ically for the distortions produced by two unstacked,
solvent-exposed bases. Our studies of modified deletion
duplexes show that sufficient stabilizing stacking inter-
actions between the bulky aromatic lesions with the
DNA bases can overcome the effects of the local distor-
tions/destabilizations caused by bulky lesions; these
stabilizing stacking interactions may hinder the insertion
of the b-hairpin and the subsequent NER steps.
A pre-flipped base from the complementary strand is not
absolutely required for lesion recognition
The full duplexes with cis-B[a]P-dG lesions have a flipped
out cytosine partner base in the complementary strand
Nucleic Acids Research, 2011,Vol.39, No. 208761
(34). It has been argued that this flipped out base plays a
key role (13) in the efficient removal of this lesion by the
NER apparatus in human cell extracts, since the absence
of this cytidine residue in deletion duplexes abolishes NER
(13,15). Yet, we show here that, even with the partner
nucleotide absent, the topological features of a lesion
can provide sufficient destabilization to, presumably,
permit b-hairpin insertion and NER. This is demonstrated
with the C8-dG-PhIP-modified deletion duplex, where
stacking is more modest than for the cis-B[a]P-dG
adduct, and steric hindrance imposed by the dynamic
non-planar phenyl ring causes neighboring base pair per-
turbations. While the NER of the C8-dG-PhIP-modified
deletion duplex is significantly smaller than that of the
C8-dG-PhIP-modified full duplex, it still exhibits signifi-
cant NER (Figure 4) even though the partner dCp is
missing entirely in the complementary strand. We
suggest that the ease of flipping the partner base out of
the duplex, which is determined by the local thermo-
dynamic destabilization, rather than the existence of a
pre-flipped base, governs b-hairpin intrusion. Strong
base stacking interactions between lesions with aromatic
ring systems and neighboring base pairs can prevent not
only the insertion of the b-hairpin between the two
strands, but also impede the flippability of the bases in
the complementary strand, an area we are currently
investigating (57). Therefore, the recognition of a bulky
lesion is a function of a combination of destabilizing
effects and stabilizing vdW interactions that depend on
the structural features of the lesions; these together deter-
mine the local thermodynamic properties of the modified
duplexes. Hence, the presence of a pre-flipped partner nu-
cleotide in the complementary strand is not an absolute
requirement for NER.
Thermodynamic stabilization of a damaged DNA duplex
through effective carcinogen–base stacking interactions
can overcome the impact of destabilizing distortions
imposed by the lesion, and thus convey resistance to
NER. The presence of the normal dCp in the complemen-
tary strand opposite the lesion exerts a destabilizing effect,
hindering the development of optimal aromatic lesion–
base stacking interactions seen in the deletion duplexes
and thus favoring efficient NER. However, the existence
of the pre-flipped dCp partner nucleotide is not an essen-
tial requisite for the initiation of NER.
Supplementary Data are available at NAR Online.
The authors gratefully acknowledge TeraGrid resources
provided by the Texas Advanced Computing Center sup-
ported by the National Science Foundation. D.A.R.,
M.K. and K.K. carried out the NER experiments.
D.A.R., A.K., M.K. and Y.C. collaborated on the
thermal melting experiments. D.A.R., A.K. and M.K.
synthesized the adducts utilizing diolepoxide and the
N-acetoxy-PhIP reactive intermediates generated by J.K.
and S.A. The computer modeling was carried out by H.M.
with the help of Y.C. and S.D. The B[a]P-DNA adduct
NMR studies were performed in the laboratory of D.J.P,
while S.B. supervised the modeling studies, and N.E.G.
supervised the experimental work. N.E.G., S.B. and
H.M. analyzed the results. N.E.G., H.M., S.B. and
D.J.P. wrote the manuscript.
National Institutes of Health (grant CA-099194 to
N.E.G., CA-75449 and CA-28038 to S.B.); (CA-046533
to D.J.P.). Components of this work were conducted in
the Shared Instrumentation Facility at NYU that was
constructed with support from a Research Facilities
Improvement (grant C06 RR-16572) from the National
Center for Research Resources, National Institutes of
Health. The acquisition of the MALDI-TOF mass spec-
trometer used in this work was supported by the National
Science Foundation (CHE-0958457). Funding for open
access charge: Grants from the National Institutes of
Health CA-099194 to N.E.G., CA-75449 and CA-28038
Conflict of interest statement. None declared.
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