Published online April 30, 2004
2474±2481 Nucleic Acids Research, 2004, Vol. 32, No. 8
DOI: 10.1093/nar/gkh568
NMR structure of the DNA decamer duplex
containing double T´G mismatches of cis±syn
cyclobutane pyrimidine dimer: implications for DNA
damage recognition by the XPC±hHR23B complex
Joon-Hwa Lee, Chin-Ju Park, Jae-Sun Shin, Takahisa Ikegami1, Hideo Akutsu1 and
Byong-Seok Choi*
Department of Chemistry and National Creative Research Initiative Center, Korea Advanced Institute of Science
and Technology, 373±1, Guseong-dong, Yuseong-gu, Daejon 305±701, Korea and 1Institute for Protein Research,
Osaka University, 3±2 Yamadaoka, Suita, 565±0871 Japan
Received March 17, 2004; Accepted April 6, 2004
ABSTRACT
The cis±syn cyclobutane pyrimidine dimer (CPD) is
a cytotoxic, mutagenic and carcinogenic DNA
photoproduct and is repaired by the nucleotide excision repair (NER) pathway in mammalian cells. The
XPC±hHR23B complex as the initiator of global
genomic NER binds to sites of certain kinds of DNA
damage. Although CPDs are rarely recognized by
the XPC±hHR23B complex, the presence of mismatched bases opposite a CPD signi®cantly
increased the binding af®nity of the XPC±hHR23B
complex to the CPD. In order to decipher the properties of the DNA structures that determine the binding af®nity for XPC±hHR23B to DNA, we carried out
structural analyses of the various types of CPDs by
NMR spectroscopy. The DNA duplex which contains
a single 3¢ T´G wobble pair in a CPD (CPD/GA
duplex) induces little conformational distortion.
However, severe distortion of the helical conformation occurs when a CPD contains double T´G
wobble pairs (CPD/GG duplex) even though the
T residues of the CPD form stable hydrogen bonds
with the opposite G residues. The helical bending
angle of the CPD/GG duplex was larger than those
of the CPD/GA duplex and properly matched
CPD/AA duplex. The ¯uctuation of the backbone
conformation and signi®cant changes in the widths
of the major and minor grooves at the double T´G
wobble paired site were also observed in the
CPD/GG duplex. These structural features were also
found in a duplex that contains the (6±4) adduct,
which is ef®ciently recognized by the XPC±hHR23B
complex. Thus, we suggest that the unique structural features of the DNA double helix (that is,
helical bending, ¯exible backbone conformation,
and signi®cant changes of the major and/or minor
grooves) might be important factors in determining
the binding af®nity of the XPC±hHR23B complex to
DNA.
INTRODUCTION
The cis±syn cyclobutane pyrimidine dimer (CPD) (Fig. 1A)
and the pyrimidine(6±4)pyrimidone photoproduct [(6±4)PP]
constitute the two major classes of cytotoxic, mutagenic and
carcinogenic DNA photoproducts induced by ultraviolet (UV)
irradiation (1±3). The CPD lesion is repaired by various kinds
of DNA repair enzymes (4±7). The T4 endonuclease V (endo
V) is a repair enzyme that catalyzes the ®rst step of the CPDspeci®c base excision repair pathway (6). The Escherichia coli
CPD-speci®c photolyase binds speci®cally to the CPD and,
upon excitation by blue light, splits the cyclobutane ring and
restores the intact base (7). In in vitro assays, the CPD among
DNA photoproducts is recognized with the lowest af®nity by
the E.coli uvrA protein, which initiates repair by the uvr(A)BC
excinuclease complex (4). Therefore, this type of lesion is
repaired about nine times more slowly by the uvr(A)BC
excinuclease than is the (6±4)PP (8).
Various structural studies of CPD-containing DNA
duplexes have suggested mechanisms of binding of the repair
proteins to CPDs (6,9±12). The crystal structure of the CPD±
endo V complex reveals that the DNA duplex exhibits a sharp
kink at the central CPD site (6). This kink causes the adenine
base opposite the 5¢ T of the CPD to ¯ip out of the DNA
duplex and be trapped in a pocket of the endo V enzyme (6).
Results of a structural study of a DNA dodecamer duplex that
contained a CPD suggest that the unique BII type backbone
*To whom correspondence should be addressed. Tel: +82 42 869 2828; Fax: +82 42 869 2810; Email: [email protected]
Present address:
Joon-Hwa Lee, Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA
The authors wish it to be known that, in their opinion, the ®rst two authors should be regarded as joint First Authors
Nucleic Acids Research, Vol. 32 No. 8 ã Oxford University Press 2004; all rights reserved
Nucleic Acids Research, 2004, Vol. 32, No. 8
Figure 1. Chemical structures analyzed in this study. (A) Chemical
structure of the CPD lesion. (B) DNA sequence contexts of the CPD/GA,
CPD/GG and CPD/AA duplexes.
2475
by the double T´G mismatches of the CPD and that additional
factors and steps might be required for CPD recognition (19).
In order to elucidate how the T´G mismatches of the CPD
affect the binding af®nity of XPC±hHR23B to a CPDcontaining DNA duplex, we determined the solution structures
of two DNA decamer duplexes. One contained a mismatched
base pair between the 3¢ T of the CPD and the opposite G
residue (referred to as the CPD/GA duplex; PDB accession no.
1pib), and the other contained two mismatched base pairs
between two consecutive T residues that are part of a CPD and
their opposing G residues (referred to as the CPD/GG duplex;
PDB accession no. 1snh). These structures were then
compared with that of a properly matched CPD (the
CPD/AA duplex), which was described in our previous
study (9). The structural differences among the various
duplexes observed in this study can account for the observed
enhancement of the XPC±hHR23B complex binding af®nity
for CPDs that contain double T´G mismatches. This structural
comparison thus provides insight into the mechanism of the
damage recognition step initiated by XPC±hHR23B during
NER.
MATERIALS AND METHODS
linkage that ¯anks the 3¢ side of the dimer might be an
important structural element for recognition of the dimer by
the CPD-speci®c photolyase and endo V repair enzymes (10).
Structural comparisons between DNA duplexes that contain a
site-speci®c CPD and a (6±4)PP suggest that the binding
af®nities of non-speci®c repair proteins, such as E.coli uvrA,
for the CPD and (6±4)PP positively correlate with the amount
of helical bending caused by the DNA damage (9).
In mammalian cells, nucleotide excision repair (NER) is the
major pathway for removal of DNA photoproducts induced by
UV light (13±15). The NER pathway is initiated by the
detection of a single damaged site among an extensive
background of undamaged DNA, which is accomplished via
two distinct mechanisms, global genomic repair (GGR) and
transcription-coupled repair (TCR). In GGR pathway, a helixdistorting DNA lesion is bound by initial damage recognition
proteins that recruit other repair proteins to the damaged site.
In human cells, the binding af®nities of the DNA damage
recognition proteins [replication protein A (RPA), XPA and
XPE] for CPDs are much lower than those for (6±4)PPs (8,16).
Recently, it was suggested that in GGR, XPC±hHR23B is
the primary damage recognition protein that initiates the
human NER pathway through binding to the damaged site
(17,18). XPC±hHR23B binds preferentially to bubble structures and bulky damaged DNA (17,19). It was also reported
that CPDs are recognized poorly by XPC±hHR23B and other
known repair proteins (19). However, under conditions in
which XPC±hHR23B hardly bound to the properly paired
CPD, the presence of double-mismatched bases opposite a
CPD signi®cantly enhances XPC±hHR23B binding whereas a
single-mismatched CPD shows marginally detectable binding
activity. Cell-free excisions of mismatched CPDs are much
more ef®cient than expected from the binding af®nity for
XPC±hHR23B (19). From these results, Sugasawa et al.
suggested that the enhancement of XPC±hHR23B binding
af®nity results from the increased structural distortion caused
Sample preparation
The CPD-containing DNA decamer was prepared by direct
254-nm UV irradiation of a DNA oligomer in an aqueous
solution and puri®ed as described (11). The CPD/GA and
CPD/GG duplexes (Fig. 1B) were prepared by dissolving the
lesion-containing DNA strands and the complementary
strands at a 1:1 stoichiometric ratio in an aqueous solution
containing 20 mM sodium phosphate (pH 7.0) and 100 mM
NaCl.
NMR experiments
All NMR data sets generated with the CPD/GA and CPD/GG
duplexes were collected with a Varian Inova 600 MHz
spectrometer (KAIST, Daejon). Details of the NMR experiments and data processing can be found in our earlier
published studies of a photoproduct-containing DNA duplex
(11,20,21). Nuclear Overhauser effect (NOE) distance restraints from nonexchangeable protons were obtained from
two-dimensional NOE spectroscopy (NOESY) experiments
with mixing times of 80, 150 and 300 ms in a D2O buffer
solution. Exchangeable proton NOEs were determined using
NOESY spectra in H2O buffer with 100 and 300 ms mixing
times. Watson±Crick-type hydrogen bonding restraints were
imposed on each base pair, except the T5±Y16 and T6±X15
base pairs. The torsion angle restraints were derived by
analyzing the data from DQF-COSY (22). Other backbone
torsion angle restraints in the ¯anking C±G base pair region
were equivalent to the values of a normal B-form helix.
Torsion angle restraints in the CPD lesion and opposite
nucleotides were only from the experimental data.
Scalar 1JCH and dipolar 1DCH couplings of the CPD/GG
duplex were derived from natural abundance sensitivity
enhancement 1H-13C HSQC experiments with and without
Pf1 (~15 mg/ml) at 25°C (see table 1 in Supplementary
Material). The data were collected on a Bruker 800 MHz DRX
spectrometer equipped with a 1H {13C, 15N} triple resonance
2476
Nucleic Acids Research, 2004, Vol. 32, No. 8
cryoprobe (Osaka University, Osaka). Pf1 ®lamentous bacteriophage was purchased from ASLA, Ltd.
Structure calculation
The structures of the CPD/GA and CPD/GG duplexes were
initially calculated using the program X-PLOR 3.1 (23) with
restrained molecular dynamics. We initially generated the
normal A- and B-form starting structures with modi®cation of
the CPD at the T5±T6 position. These structures were
subjected to restrained molecular dynamics and simulated
annealing protocols. Details of the structure calculation
protocols can be found in our earlier published studies of a
photoproduct-containing DNA duplex (15,20,21). Fifteen
structures of the CPD/GA duplex and 13 structures of the
CPD/GG duplex were chosen on the basis of the lowest total
energies.
The ensemble of the CPD/GG duplex was then re®ned with
RDCs in addition to NOE and torsion angle restraints. The
calculation was performed by XPLOR±NIH version (24).
Alignment tensor analysis of the observed residual dipolar
coupling was performed as before (25,26). The values Da = ±
13.0 Hz and R = 0.4 were used in the calculation. The
re®nement step consists of an initial equilibration stage where
the dipolar coupling force constants were increased from 0.01
to 5.0 kcal mol±1 Hz±2 over 50 cycles, corresponding to a 15 ps
molecular dynamics run. This was followed by a 20 ps
restrained molecular dynamics calculation at 300 K. The ®nal
structures were generated after 2000 cycles of energy
minimization (25).
The helical parameters of the re®ned structures were
calculated using the program CURVES 5.3 (27) and Figures
2 and 7 were prepared with the program MOLMOL (28).
RESULTS AND DISCUSSION
Overall features of the solution structures of the CPD/
GA and CPD/GG duplexes
In the CPD/GA duplex, a converged subset of 15 structures
was identi®ed on the basis of low NOE violations and total
energies. These structures exhibited pairwise root-meanÊ for all
squared deviation (r.m.s.d.) values of 1.05 6 0.24 A
heavy atoms. The 13 superimposed re®ned structures of the
CPD/GG duplex exhibited pairwise r.m.s.d. values of 1.10 6
Ê for all heavy atoms (Table 1).
0.43 A
Figure 2A and B shows stereo views of the mean structures
of the CPD/GA and CPD/GG duplexes, respectively. Views of
individual structures superimposed on the mean structures of
the CPD/GA and CPD/GG duplexes show they are reasonably
well converged (Fig. 2C). A ribbon representation was applied
to the phosphate backbone of each duplex in order to
emphasize the distortion of the phosphate backbone caused
by formation of the cyclobutane ring and the T±G wobble
pairs.
The T residues of the CPD formed stable wobble pairs
with the opposite G residues
The H6 and 5-methyl resonances of the T5 and T6 residues
were assigned from a unique NOE feature produced by four
substituents of the cyclobutane ring of the CPD (Fig. 3A). The
strong NOE cross-peak between the imino proton resonance of
Figure 2. The structures of CPD/GA and CPD/GG duplex. (A) The stereo
view of the CPD/GA duplex, and (B) of the CPD/GG duplex. In both
duplexes, the two thymine residues (T5, T6) of the CPD are colored green,
and the G15 residues are colored pink. The A16 residue in the CPD/GA
duplex and the G16 residue in the CPD/GG duplex are colored blue and
pink, respectively. (C) Left panel. Superimposed views of 15 calculated
structures of the CPD/GA duplex. Right panel. Superimposed views of 13
calculated structures of the CPD/GG duplex.
T5 and the H2 resonance of the opposite A16 in the CPD/GA
duplex indicates that the T5´A16 base pair maintains the
normal Watson±Crick T´A base pair (Fig. 4A). As was the
case for the CPD/AA duplex (9), in the CPD/GA duplex,
formation of the CPD caused some structural distortion of the
5¢ T´A base pair, which was con®rmed by the unusual values
of the propeller twist angle. The T6´G15 wobble pair of the
CPD formed hydrogen bonds between the T6-imino and G15O6 and between the G15-imino and T6-O2 (Fig. 4A). This
hydrogen bonding feature is consistent with the observation of
a strong NOE cross-peak between the two imino protons of the
T6´G15 mismatch (see ®gure 1 in Supplementary Material).
Nucleic Acids Research, 2004, Vol. 32, No. 8
Table 1. Structural statistics for CPD/GA and CPD/GG
Total number of distance restraints
Intra-residue
Inter-residue
Dihedral restraints
Base planarity restraints
Residual dipolar coupling restraints
Total number of restraints
Ê)
Pairwise r.m.s.d. for all heavy atoms (A
Ê)
Average NOE violations (A
Average dihedral angle violations (°)
Mean deviation from covalent geometry
Ê)
Bond lengths (A
Angles (°)
Impropers (°)
CPD/GA
CPD/GG (Re®ne)
501
411
90
112
10
0
623
1.05 6 0.24
0
0
500
378
122
112
10
13
635
1.10 6 0.43
0
0
0.014
2.71
5.32
0.014
2.49
5.68
2477
Ê ) value and, thus, the distance between
has a short rise (~2.4 A
the methyl carbon of T5 and the C8 molecule of base A4 is
very short (Fig. 3B). This structural feature and the location of
the T5-methyl group can explain the unusual up®eld-shift of
the T5-methyl resonance, which is caused by a shielding effect
of the A4 base ring current.
In contrast, the T5-methyl resonance in the CPD/GG duplex
was signi®cantly down®eld-shifted to 1.33 p.p.m. (Fig. 3A). In
the CPD/GG duplex, the A4/T5 base step feature is signi®cantly different from that of the CPD/GA duplex (Fig. 3B and
C). In the CPD/GG duplex, the T5´G16 wobble pair changed
Ê ) and the location
the rise value of the A4/T5 base step (~2.9 A
of the T5-methyl (Fig. 3C). This structural change disrupted
the shielding effect of the A4 base on the T5-methyl
resonance, as was evident by the down®eld shift of the T5methyl resonance (Fig. 3A). In the CPD/GG duplex, the base
stacking interaction at the CPD site and 3¢ ¯anking region was
disrupted, as evidenced by its negative roll (~±24°) angle; this
observation is similar to that made for a DNA duplex decamer
that contained a 3¢ T´T base pair in its CPD (15) and in contrast
to the CPD/GA duplex, which was well stacked. This indicates
that the 5¢ T´G wobble pair of the CPD causes structural
distortions not only in the 5¢ ¯anking region, but also in the 3¢
¯anking region of the CPD.
The calculated structure of the CPD/GA duplex showed that
the T6´G15 base pair was slightly distorted, as con®rmed by
the propeller twist (~24°) value.
In the CPD/GG duplex, the two imino proton resonances of
the T5 and T6 residues showed strong NOEs with the imino
protons of the opposite G16 and G15 residues, respectively.
The two T (T5, T6) residues of the CPD in the CPD/GG
duplex formed wobble base pairs with the opposite G residues,
similar to the T6´G15 base pair in the CPD/GA duplex
(Fig. 4B). However, the calculated structure of the CPD/GG
duplex showed that the T6´G15 base pair was distorted more
severely than that of CPD/GA as con®rmed by the unusual
propeller twist (~24°) and buckle (~±44°) angles. The T5´G16
wobble pair was also distorted severely; it showed a large
negative propeller twist value.
In temperature-dependent imino proton spectra conducted
in H2O buffer (Fig. 5), all imino resonances except those of the
terminal base pairs in both duplexes were intact over 25°C,
indicating that the CPD/GG and CPD/GA duplexes form
stable double helices at room temperature. However, the
melting temperature of the CPD/GG duplex was 10°C lower
than that of the CPD/GA duplex (Fig. 5). This indicates that
the double T´G mismatches of the CPD slightly destabilize the
DNA double helix. In both duplexes, the imino proton
resonances of the mismatched T and G residues located at
the central CPD site show that these residues have the same
melting temperature as that of the overall helix. This means
that the T residues of the CPD form stable base pairs with the
opposite G residues rather than bubble-like structures which
may be caused by weak base pairing.
In the previous study, we found that formation of the CPD
caused a slight bending of the DNA helix in the CPD/AA
duplex (9). Our structural calculation herein showed that the 3¢
T´G mismatch of the CPD maintained this helical bending
(~10°) in the CPD/GA duplex. However, more helical bending
was observed in the calculated structures of CPD/GG duplex.
Recently, it was found by scanning force spectroscopy that the
XPC±hHR23B complex induces a bend in the DNA helix
upon binding and that this bend stabilized XPC±hHR23B
binding at the damaged site (29). It was also reported that the
helical bending angle caused by a (6±4)PP lesion, which is a
good substrate for binding to the XPC±hHR23B complex, is
quite large (~44°) (9,30). Unusual DNA structures, such as
those that contain bubbles and junctions between the singleand double-strands, yield ¯exible helices that are easily bent
by the binding of a protein. DNA damage and unusual DNA
structures, which are ef®ciently recognized by the XPC±
hHR23B complex, appear to have the helical bending
properties or allow helices to be bent easily at the binding
site of a protein complex. Thus, we hypothesize that the
helical bending properties observed in the CPD/GG duplex
may enhance the binding af®nity of the XPC±hHR23B
complex for the mismatched CPD lesion.
Base stacking interactions at the CPD sites differ
between the two duplexes
The backbone conformation of the CPD/GG duplex is
distorted and ¯exible
We also studied the base stacking interactions at the CPD sites
and the 5¢ and 3¢ ¯anking regions of the CPD/GA and CPD/
GG duplexes. The purine bases (A16 in CPD/GA and G16 in
CPD/GG) opposite the 5¢ T (T5) of the CPD were nicely
stacked with the 5¢ ¯anking T17 bases (Fig. 3B and C). The
chemical shift (0.56 p.p.m.) of the T5-methyl resonance in the
CPD/GA duplex was similar to that of the CPD/AA duplex
(0.57 p.p.m.) (Fig. 3A). As was the case with the CPD/AA
duplex, structure calculation revealed that the A4/T5 base step
In both structures, the backbone conformation involving the
phosphorous atom between the 3¢ T (T6) of the CPD and its 3¢
neighboring A7 residue is distorted, as con®rmed by the
gauche± orientation of the e angle and the trans orientation of
the z angle in the calculated structures. Because H2¢±H3¢,
H2"±H3¢ and H3¢±H4¢ cross-peaks of T6 of both duplexes are
absent in the DQF-COSY spectra (data not shown), we can
deduce that the e angles of T6 bases are in either the trans or
gauche± region (SJH3¢ < 10 Hz) (31). Even though we did not
The CPD/GG duplex exhibits signi®cant helical bending
2478
Nucleic Acids Research, 2004, Vol. 32, No. 8
Figure 3. (A) NOE cross-peaks between the H6 and methyl protons of the two T residues of the CPD in the expanded NOESY spectra in a D2O buffer at
17°C. The spectrum of the CPD/GA duplex is colored blue and that of the CPD/GG duplex is colored red. (B) Comparative stick views (top: front view,
bottom: top view) of the stacking interactions between the A4´T17 and T5´A16 base pairs in the CPD/GA duplex, and (C) between the A4´T17 and T5´G16
base pairs in the CPD/GG duplex.
Figure 4. (A) Ball and stick views of the T5´A16 and T6´G15 base pairs in
the CPD/GA duplex, and (B) of the T5´G16 and T6´G15 base pairs in the
CPD/GG duplex. Balls are colored using the accepted atomic color
representation: gray, hydrogen; green, carbon; blue, nitrogen; red, oxygen.
The dotted lines indicate the hydrogen bonds determined by the program
INSIGHT II.
put any arti®cial dihedral restraints on these damaged bases
and opposite ones, the calculated structures showed the e
angles of T6 bases had gauche± conformations. This BII
conformation is in accord with the analysis of scalar coupling
patterns and is expected to play a crucial role in the
recognition of a CPD by repair enzyme, such as endo V (10).
We also found that some of the calculated backbone torsion
angles for the CPD/GG duplex are not within a single
conformational range. This applies mainly to the damaged
bases and their neighbors. In the calculated structures, the g
angles of G15 and G16 were measured in either the gauche+ or
the trans range. There were no H3¢±H4¢, H4¢±H5¢ and H4¢±
H5" cross-peaks of G15 and G16 in the DQF-COSY spectra,
which implies that the g angles of both bases may be in either
the gauche+ or the anti± range. However, the possibility of the
anti± range can be excluded by analyzing NOESY data
obtained at 1°C (31). This means that the g angles of G15 and
G16 may have the gauche+ conformation at least at the low
temperature. Because the sequential NOE cross-peaks and
even the intra-NOE cross-peaks of G15 between base and
sugar at these steps were not observed or were very weak in
NOESY spectra recorded at 17°C, whereas these peaks exist in
NOESY spectra recorded at 1°C, it is expected that the
residues may experience a kind of slow dynamical process
(Fig. 6). Although the proposal of a ¯exible DNA backbone
from experimental data and calculation results is a reasonable
one, it is rather dangerous to propose this ¯exibility solely on
the basis of observed torsion angles inter-conversion in the
course of restrained molecular dynamics. Additionally, the
previous simulation study also showed that the phosphate that
bridges the bases complementary to the CPD had ¯exibilities
in the CPD/AA duplex (32).
Even though the sugar-backbone conformations at these
step are somewhat ¯exible in nature, the structural conformations of these base pairs are well converged in the ensemble
of our calculated structures, as evidenced by strong imino
resonances in the temperature-dependent 1-D spectra (Fig. 5).
It was reported that the XPC±hHR23B complex shows
higher af®nity for single-stranded DNA than for doublestranded DNA (33). Also, certain secondary DNA structures
were strongly preferred by the complex, such as single- and
double-stranded junctions (33) and three- or ®ve-nucleotide
bubble structures (19). However, Batty et al. clearly showed
single-stranded character by itself was an insuf®cient explanation for binding of XPC±hHR23B complex to DNA by the
competition experiments (34). These results imply that the
DNA-binding af®nity of the XPC±hHR23B complex may
correlate with the presence of the ¯exibility provided by the
unpaired bases in a DNA double helix. In the three CPDcontaining DNA duplexes we studied here, all T residues of
the CPDs formed stable hydrogen bonds with the opposite A
or G residues. However, even though the two T residues of the
CPD in the CPD/GG duplex were able to form stable wobble
Nucleic Acids Research, 2004, Vol. 32, No. 8
2479
Figure 5. Temperature dependence of the imino proton resonances of the 1H-NMR spectra for the CPD/GA (left) and CPD/GG (right) duplexes in an H2O
buffer solution. The positions of nucleotides in the decamer duplex that give rise to the resonances are indicated. The experimental temperatures are shown at
the center of the ®gure.
single- and double-strands are extremely ¯exible in the singlestranded regions. The (6±4)PP lesion also has a ¯exible
backbone conformation at its 3¢ T site and melts at room
temperature (13,30). Thus, we suggest that the ¯exibility of
the distorted backbone conformation of double-stranded DNA
may be an important factor in determining the binding af®nity
of the XPC±hHR23B complex for DNA.
Widening of the major groove and narrowing of the
minor groove at the CPD site in the CPD/GG duplex
Figure 6. Comparison of the expanded NOESY spectra of the CPD/GG duplex at 17°C (blue) and at 1°C (red). Selective resonances are labeled using
the same color as the spectra. Resonances that overlapped in both spectra
are shown in black.
pairs with the opposite G residues, the binding af®nity of the
XPC±hHR23B complex for this CPD and the repair rate for
this CPD via NER were increased signi®cantly. These ®ndings
imply that the XPC±hHR23B complex does not recognize
unpaired bases directly, but, rather, recognizes the structural
distortion caused by bases that are unpaired or mismatched.
We mentioned that the CPD/GG duplex has a highly ¯exible
backbone conformation, which is consistent with the fact that
we observed no sequential NOE cross-peaks at the CPDdamaged site at 17°C (Fig. 6). The backbone conformations
of DNA structures that have bubbles or junctions between
Figure 7 shows the major and minor grooves of the three DNA
duplex decamers that contained a CPD and a (6±4)PPcontaining DNA duplex decamer with the same ¯anking
sequences. The CPD caused widening of the minor groove and
narrowing of the major groove at the damage site in both the
CPD/AA and CPD/GA duplexes (Fig. 7). This is consistent
with the result from the crystal structure of CPD±endo V
complex which shows that the protein interacts with the DNA
in the minor groove (6). In contrast, for the CPD/GG duplex,
the major groove at the CPD site was signi®cantly wider than
that of an average B-DNA structure (Fig. 7C). In addition, the
minor groove at the CPD site of the CPD/GG duplex narrowed
Ê (Fig. 7G). The groove width of a DNA double
greatly to 2~4 A
helix can play a crucial role in the formation of DNA±protein
complexes, and it has been suggested that the change in
groove width in¯uences the speci®c binding af®nity of
proteins for CPDs in the context of the genome (6). As with
the CPD/GG duplex, a (6±4)PP caused the enlargement of the
major groove and compression of the minor groove at the
damaged site (Fig. 7D and H). It is possible that this signi®cant
change in groove width aids the XPC±hHR23B complex in
detection of the CPD site. However, the major and minor
groove widths of CPD in the CPD/GG duplex are less deviated
from the normal B-DNA than that of (6±4)PP.
2480
Nucleic Acids Research, 2004, Vol. 32, No. 8
Figure 7. Electrostatic potential surface models. Views of the major grooves of (A) CPD/AA, (B) CPD/GA, (C) CPD/GG, and (D) the (6±4)PP-containing
decamer duplex. Views of the minor grooves of (E) CPD/AA, (F) CPD/GA, (G) CPD/GG and (H) the (6±4)PP-containing decamer duplex. The damaged
sites and residues opposite the damage are indicated by arrows and labeled.
Implications for DNA damage recognition by the
XPC±hHR23B complex
CPDs are produced directly by sunlight at a 5 to 10 times
higher rate than the other major photoproduct, (6±4)PP (2,3).
In the NER pathway, most DNA damage binding proteins
show lower binding af®nities for CPD than for other DNA
photoproducts (4,12). The XPC±hHR23B complex, which is
involved in the DNA damage recognition step in human cells,
also rarely binds to CPD lesions (17,19). Thus, because of its
high abundance and low repair rate, the CPD might be the
most mutagenic photoproduct in eukaryotic cells. One unique
feature of the NER pathway is that the XPC±hHR23B
complex binds to a variety of DNA lesions and unusual
structures with different af®nities (1±3).
The XPC±hHR23B complex strongly prefers to bind to
single-stranded DNA, speci®cally, secondary DNA structures
that contain a single- and double-stranded junction and threeor ®ve-nucleotide bubble structures (19,33). The complex also
shows high binding af®nity for DNA duplexes that contain
(6±4)PP lesions (17). DNA photoproducts, base mismatches,
and single-stranded regions all destabilize the DNA double
helix and thus decrease the melting temperature of the DNA
duplex (35,36).
Sugasawa et al. found that double T´G wobble pairs in
CPDs enhance the binding af®nity of the XPC±hHR23B
complex for the CPD (19). It was believed that an understanding of the relationship between this enhanced binding
af®nity and the structural features induced by the double T´G
wobble pairs would lead to a deciphering of the molecular
mechanisms behind the damage recognition step initiated by
the XPC±hHR23B complex. In the CPD/GA duplex, the single
G residue, which forms stable hydrogen bonds with the
opposite 3¢ T residues of the CPD, causes a slightly distorted
backbone conformation similar to that of the CPD duplex with
two A residues. However, having double T´G wobble pairs in
the CPD caused not only an unusual base pair geometry, but
also backbone distortion, even though the two T residues of
the CPD lesion formed stable hydrogen bonds with the
opposite G residues (CPD/GG duplex). Among these structural features, we found three distortions related to the DNA
backbone in the CPD/GG duplex that may correlate with
binding of the XPC±hHR23B complex to DNA. First, the
widths of the major and minor grooves in the CPD/GG duplex
were greatly widened and narrowed, respectively, in contrast
to the CPD/AA and CPD/GA duplexes. Second, the double
T´G wobble pairs of the CPD formed stable hydrogen bonding,
but induced ¯exibility into the sugar-backbone conformation
of the DNA helix. The third feature is the helical bending
caused by the double T´G wobble pairs in the CPD/GG duplex,
which is more severe than those observed in CPD/AA and
CPD/GA duplexes. This structural distortion may correlate
with the greater af®nity of the XPC±hHR23B complex for the
CPD in the CPD/GG duplex, versus that in duplexes with
matched or single T´G mismatched base pairs.
As discussed above, these structural features can be found
in DNA duplexes that contain a (6±4)PP, which is the best
DNA substrate for the XPC±hHR23B complex among the
various DNA photoproducts. Some ¯exible DNA structures
such as three- or ®ve-nucleotide bubbles and partially singlestranded DNA duplexes, which are good substrates for the
XPC±hHR23B complex, were able to obtain these structural
features easily after binding to the complex. Thus, we suggest
that the XPC±hHR23B complex recognizes DNA damage by
searching for unusual groove width, ¯exible backbone distortion, and/or a bent helix during damage recognition in NER.
Nucleic Acids Research, 2004, Vol. 32, No. 8
Coordinates
The atomic coordinates and experimental NMR restraints of
the CPD/GA and CPD/GG duplexes have been deposited in
the Protein Data Bank, www.rscb.org [PDB ID code: 1pib
(CPD/GA), 1snh (CPD/GG)].
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We thank Dr Sung-Hun Bae for useful discussions. This work
was supported by the Creative Research Initiative Program
from the Ministry of Science and Technology, Korea. C.-J.P.
and J.-S.S. were supported partially by the BK21 project.
REFERENCES
1. Patrick,M.H. and Rahn,R.O. (1976) Photochemistry of DNA and
polynucleotides: Photoproducts. In Wang,S.Y. (ed.), Photochemistry and
Photobiology of Nucleic Acids. Academic Press, New York, Vol. II, pp.
35±95.
2. Mitchell,D.L. (1988) The relative cytotoxicity of (6±4) photoproducts
and cyclobutane dimers in mammalian cells. Photochem. Photobiol., 48,
51±57.
3. Brash,D.E. (1988) UV mutagenic photoproducts in Escherichia coli and
human cells: a molecular genetics perspective on human skin cancer.
Photochem. Photobiol., 48, 59±66.
4. Svoboda,D.L., Smith,C.A., Taylor,J.-S. and Sancar,A. (1993) Effect of
sequence, adduct type and opposing lesions on the binding and repair of
ultraviolet photodamage by DNA photolyase and (A)BC excinuclease.
J. Biol. Chem., 268, 10694±10700.
5. Sancar,A. (1994) Structure and function of DNA photolyase.
Biochemistry, 33, 2±9.
6. Vassylyev,D.G., Kashiwagi,T., Mikami,Y., Ariyoshi,M., Iwai,S.,
Ohtsuka,E. and Morikawa,K. (1995) Atomic model of a pyrimidine
dimer excision repair enzyme complexed with a DNA substrate:
structural basis for damaged DNA recognition. Cell, 83, 773±782.
7. Park,H.-W., Kim,S.-T., Sancar,A. and Deisenhofer,J. (1995) Crystal
structure of DNA photolyase from Escherichia coli. Science, 268,
1866±1872.
8. Reardon,J.T., Nichols,A.F., Keeney,S., Smith,C.A., Taylor,J.-S., Linn,S.
and Sancar,A. (1993) Comparative analysis of binding of human
damaged DNA-binding protein (XPE) and Escherichia coli damage
recognition protein (UvrA) to the major ultraviolet photoproducts:
T[c,s]T, T[t,s]T, T[6-4]T and T[Dewar]T. J. Biol. Chem., 268,
21301±21308.
9. Kim,J.-K., Patel,D.J. and Choi,B.-S. (1995) Contrasting structural
impacts induced by cis-syn cyclobutane dimer and (6±4) adduct in DNA
duplex decamers: implication in mutagenesis and repair activity.
Photochem. Photobiol., 62, 44±50.
10. McAteer,K., Jing,Y., Kao,J., Taylor,J.-S. and Kennedy,M.A. (1998)
Solution-state structure of a DNA dodecamer duplex containing a Cis-syn
thymine cyclobutane dimer, the major UV photoproduct of DNA. J. Mol.
Biol., 282, 1013±1032.
11. Lee,J.-H., Choi,Y.-J. and Choi,B.-S. (2000) Solution structure of the
DNA decamer duplex containing a 3¢-T x T basepair of the cis-syn
cyclobutane pyrimidine dimer: implication for the mutagenic property of
the cis-syn dimer. Nucleic Acids Res., 28, 1794±1801.
12. Park,H.-J., Zhang,K., Ren,Y., Nadji,S., Sinha,N., Taylor,J.-S. and
Kang,C.-H. (2002) Crystal structure of a DNA decamer containing a cissyn thymine dimer. Proc. Natl Acad. Sci. USA, 99, 15965±15970.
13. Wood,R.D. (1999) DNA damage recognition during nucleotide excision
repair in mammalian cells. Biochimie, 81, 39±44.
14. deLaat,W.L., Jaspers,N.G. and Hoeijmakers,J.H. (1999) Molecular
mechanism of nucleotide excision repair. Genes Dev., 13, 768±785.
2481
15. Batty,D.P. and Wood,R.D. (2000) Damage recognition in nucleotide
excision repair of DNA. Gene, 241, 193±204.
16. Burns,J.L., Guzder,S.N., Sung,P., Prakash,S. and Prakash,L. (1996) An
af®nity of human replication protein A for ultraviolet-damaged DNA.
J. Biol. Chem., 271, 11607±11610.
17. Sugasawa,K., Ng,J.M.Y., Masutani,C., Iwai,S., van der Spek,P.J.,
Eker,A.P.M., Hanaoka,F., Bootsma,D. and Hoeijmakers,J.H. (1998)
Xeroderma pigmentosum group C protein complex is the initiator of
global genome nucleotide excision repair. Mol. Cell, 2, 223±232.
18. Lindahl,T. and Wood,R.D. (1999) Quality control by DNA repair.
Science, 286, 1897±1905.
19. Sugasawa,K., Okamoto,T., Shimizu,Y., Masutani,C., Iwai,S. and
Hanaoka,F. (2001) A multistep damage recognition mechanism for
global genomic nucleotide excision repair. Genes Dev., 15, 507±521.
20. Lee,J.-H., Hwang,G.-S. and Choi,B.-S. (1999) Solution structure of a
DNA decamer duplex containing the stable 3¢ T.G base pair of the
pyrimidine(6±4)pyrimidone photoproduct [(6±4) adduct]: implications
for the highly speci®c 3¢ T ® C transition of the (6±4) adduct. Proc. Natl
Acad. Sci. USA, 96, 6632±6636.
21. Lee,J.-H., Bae,S.-H. and Choi,B.-S. (2000) The Dewar photoproduct of
thymidylyl(3¢®5¢)-thymidine (Dewar product) exhibits mutagenic
behavior in accordance with the `A rule'. Proc. Natl Acad. Sci. USA, 97,
4591±4596.
22. Allawi,H.T. and SantaLucia,J.,Jr (1998) NMR solution structure of a
DNA dodecamer containing single G.T mismatches. Nucleic Acids Res.,
26, 4925±4934.
23. BruÈnger,A.T. (1992) X-PLOR Version 3.1: A System for X-ray
Crystallography and NMR. Yale University Press, New Haven, CT.
24. Schwieters,C.D., Kuszewski,J.J., Tjandra,N. and Clore,G.M. (2003) The
Xplor-NIH NMR molecular structure determination package. J. Magn.
Reson., 160, 66±74.
25. Tjandra,N., Tate,S., Ono,A., Kainosho,M. and Bax,A. (2000) The NMR
structure of a DNA dodecamer in an aqueous dilute liquid crystalline
phase. J. Am. Chem. Soc., 122, 6190±6200.
26. Zweckstetter,M. and Bax,A. (2000) Prediction of sterically induced
alignment in a dilute liquid crystalline phase: aid to protein structure
determination by NMR. J. Am. Chem. Soc., 122, 3791±3792.
27. Lavery,R. and Sklenar,H. (1988) De®ning the structure of irregular
nucleic acids: conventions and principles. J. Biomol. Struct. Dyn., 6,
63±91.
28. Koradi,R., Billeter,M. and WuÈthrich,K. (1996) MOLMOL: a program for
display and analysis of macromolecular structures. J. Mol. Graph., 14,
51±55.
29. Janñiijeviñ,A., Sugasawa,K., Shimizu,Y., Hanaoka,F., Wijgers,N.,
Djurica,M., Hoeijmakers,J.H.J. and Wyman,C. (2003) DNA bending by
the human damage recognition complex XPC-HR23B. DNA Repair, 2,
325±336.
30. Kim,J.-K. and Choi,B.-S. (1995) The solution structure of DNA duplexdecamer containing the (6±4) photoproduct of
thymidylyl(3¢®5¢)thymidine by NMR and relaxation matrix re®nement.
Eur. J. Biochem., 228, 849±854.
31. Kim,S.G., Lin,L.J. and Reid,B.R. (1992) Determination of nucleic acid
backbone conformation by 1H NMR. Biochemistry, 31, 3564±3574.
32. Yamaguchi,H., van Aalten,D.M., Pinak,M., Furukawa,A. and Osman,R.
(1998) Essential dynamics of DNA containing a cis.syn cyclobutane
thymine dimer lesion. Nucleic Acids Res., 26, 1939±1946.
33. Sugasawa,K., Shimizu,Y., Iwai,S. and Hanaoka,F. (2002) Molecular
mechanism for DNA damage recognition by the xeroderma pigmentosum
group C protein complex. DNA Repair, 1, 95±107.
34. Batty,D., Rapic'-Otrin,V., Levine,A.S. and Wood,R.D. (2000) Stable
binding of human XPC complex to irradiated DNA confers strong
discrimination for damaged sites. J. Mol. Biol., 300, 275±290.
35. Aboul-ela,F., Koh,D., Tinoco,Jr,I. and Martin,F.H. (1985) Base±base
mismatches. Thermodynamics of double helix formation for dCA3XA3G
+ dCT3YT3G (X, Y = A,C,G,T). Nucleic Acids Res., 13, 4811±4824.
36. Jing,Y., Kao,J.F. and Taylor,J.-S. (1998) Thermodynamic and base
pairing studies of matched and mismatched DNA dodecamer duplexes
containing cis-syn, (6±4) and Dewar photoproducts of TT. Nucleic Acids
Res., 26, 3845±3853.