Originally published In Press as doi:10.1074/jbc.M208103200 on September 17, 2002
J. Biol. Chem., Vol. 277, Issue 47, 45068-45074, November 22, 2002
Comparative Mutagenesis of the C8-Guanine Adducts
of 1-Nitropyrene and 1,6- and 1,8-Dinitropyrene in a CpG Repeat
Sequence
A SLIPPED FRAMESHIFT INTERMEDIATE MODEL FOR DINUCLEOTIDE
DELETION*,
Pablo
Hilario
,
Shixiang
Yan§,
Brian E.
Hingerty¶,
Suse
Broyde
, and
Ashis K.
Basu
**
From the
Department of Chemistry, University of
Connecticut, Storrs, Connecticut 06269, the § Department
of Chemistry and the
Department of Biology, New York University,
New York, New York 10003, and ¶ Life Sciences Research
Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee 37830
Received for publication, August 8, 2002, and in revised form, September 10, 2002
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ABSTRACT |
In the Ames Salmonella
typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and
1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP).
Genetic experiments established that certain differences in the
metabolism of the DNPs, which in turn result in increased DNA
adduction, play a role. It remained unclear, however, if the DNP
adducts, N-(guanin-8-yl)-1-amino-6 (8)-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on
the pyrene ring covalently linked to the guanine C8, are more mutagenic
than the major 1-NP adduct,
N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In
order to address this, we have compared the mutation frequency of the
three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and
Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors
containing these adducts and a control were constructed and replicated
in Escherichia coli. A remarkable difference in the induced
CpG deletion frequency between these adducts was noted. In
repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and
10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP
adducts are more mutagenic than the 1-NP adduct in the repetitive CpG
sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in
methyl-directed mismatch repair, and a dnaQ strain, which
carries a defect in proofreading activity of the DNA polymerase III.
Modeling studies showed that the nitro group on the pyrene ring at the
8-position can provide additional stabilization to the two-nucleotide
extrahelical loop in the promutagenic slipped frameshift intermediate
through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the
nitro-containing adducts compared with the Gua-C8-AP adduct itself.
 |
INTRODUCTION |
1-Nitropyrene (1-NP)1
and the dinitropyrenes are common environmental pollutants (1-3). Most
of the nitropyrenes are mutagenic (for a review see Ref. 4) and
tumorigenic (5-8), but their potency sometimes differs by more than an
order of magnitude. Nitroreduction is a major pathway of bioactivation
of all nitropyrenes, whereas O-esterification enzymes, in
addition, play a crucial role in the mutagenicity of DNPs.
1-NP and the DNPs revert Salmonella typhimurium frameshift
tester strains TA98 and TA1538 more efficiently than the strains TA100
and TA1535 that detect base pair substitutions (9). The most frequent
mutation among the revertants in TA98 is a two-base deletion of a GpC
or CpG pair within a CGCGCGCG hotspot sequence upstream of the
hisD3052 mutation (10). The frequency of reversion induced
by 1-NP in TA98 drops sharply in TA98NR that lacks the classical
nitroreductase (11). By contrast, the frequency of reversion by 1,6- and 1,8-DNP is only slightly lower in TA98NR but is significantly
reduced in TA98/1,8-DNP6, which is deficient in a specific
arylhydroxylamine esterification enzyme (11, 12). It appears that this
enzyme is necessary for the expression of mutagenicity of the DNPs but
not for that of 1-NP. The C8 guanine adducts of 1-NP and DNPs (Fig.
1) have been thought to be responsible for a major fraction of their mutagenicity. Several site-specific studies from our laboratory showed that the 1-NP adduct
N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP) is mutagenic in
Escherichia coli (13-15). However, the type and frequency
of mutations are dependent on DNA sequence context.

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Fig. 1.
Structures of the major 2'-deoxyguanosine
adducts formed by reductively activated 1-NP and the DNPs. Torsion
angles , ', and ' are defined as follows: ,
O4'-C1'-N9-C4; ', N9-C8-N(AP)-C1(AP); and ',
C8-N(AP)-C1(AP)-C10A(AP).
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Despite the acknowledged role of metabolism and the ability of each
nitropyrene to induce frameshifts, neither the frequency nor the
genetic requirements for mutagenesis of the adducts formed by these
carcinogens have ever been compared in the same organism. In order to
address such structure-activity relationships, in the current work we
have examined mutagenicity of the C8 guanine adducts formed by 1-NP and
1,6- and 1,8-DNP in a repetitive CpG sequence. We have constructed
single-stranded M13 bacteriophage genomes in which an adduct was placed
at the underscored dG of an inserted CGCGCG sequence in the
lacZ
fragment. Mutagenicity of the three adducts was
examined in strains that are either repair-competent or contained a
defect in the dnaQ gene that encodes the
-subunit of DNA
polymerase III holoenzyme. Pol III from these strains has been
shown to be defective in 3'-5'-proofreading exonuclease activity (16).
In addition, we have determined the effects of this adduct in a strain
with impaired postreplicative mismatch repair. Our results suggest
formation of an extra-helical loop as the promutagenic intermediate,
the stability of which may be linked to the mutation frequency. A
modeling study was carried out to explore the structure of this
hypothesized intermediate and to examine plausible reasons for added
stability in the case of the nitro-containing adducts. Our findings
suggest added possibilities for hydrogen bonding by the nitro group as
likely sources of added stabilization.
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EXPERIMENTAL PROCEDURES |
Materials--
E. coli strains GW5100 (JM103,
P1
), DL7 (AB1157, lac U169,
uvr+), and DL6 (uvrA), which carry a
chromosomal lac deletion, were reported (17). NR9295
(ara, thi,
prolac, F'128-27) and its derivatives NR9294
(mutS101) and NR11446
(zae::Tn10dCam, dnaQ49, zae502::Tn10, F'cc105) were provided by
R. Schaaper (NIEHS, Research Triangle Park, NC).
1-NP, 1-aminopyrene, 1-bromo-6(8)-nitropyrene,
m-chloroperoxybenzoic acid, and other chemicals for the
synthesis of 1-NOP and the nitrosonitropyrenes were from Aldrich.
Ethidium bromide and polyethylene glycol 8000 were obtained from Sigma.
M13 DNA sequencing kit, E. coli single strand binding
protein, and Sequenase version 2.0 were purchased from Amersham
Biosciences. Isopropyl
-D-thiogalactopyranoside (IPTG)
and 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside
(X-gal) were from Gold Biotechnologies (St. Louis, MO). T4
polynucleotide kinase and DNA ligase were obtained from Invitrogen.
EcoRI was from Roche Molecular Biochemicals. BssHII and exonuclease III were purchased from New England
Biolabs (Beverly, MA). [
-35S]dATP was from PerkinElmer
Life Sciences.
Methods--
Oligodeoxynucleotides were synthesized on an
Applied Biosystems, model 380B, DNA synthesizer, using the
phosphoramidite method. HPLC separations were performed using
reverse-phase columns (Phenomenex Ultracarb C-18, 4.6 × 250 mm).
Bacteriophage M13mp7L2 DNA was prepared as described (18).
Synthesis of d(CGCG*CG)--
Fifty nmol of d(CGCGCG) was stirred
at ambient temperature with 15 µmol of 1-nitrosopyrene or 1-nitroso-6
(8)-nitropyrene and 3 µmol of ascorbic acid in 100 µl of
N,N-dimethylformamide/sodium acetate/acetic acid
buffer (0.1 M), pH 5.0 (1:9), under nitrogen and protected
from light. An additional aliquot of 3 µmol of ascorbic acid was
added every 15 min during the 1st h, and the reaction was allowed to
continue for 16-20 h (19, 20). Noncovalently bound material was
removed by extensive extraction with chloroform. The adducted and
unadducted oligonucleotides were separated by reversed phase HPLC.
Further purification of the oligonucleotides was carried out by
denaturing PAGE. The oligonucleotides were desalted on a Sephadex G-10
column, dried, and stored at
20 °C until further use.
Construction of Site-specifically Modified M13
Genomes--
Bacteriophage M12mp7L2 (200 µg) was digested with a
large excess of EcoRI (2400 units) for 2 h at 25 °C
in 1 ml of 100 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 50 mM NaCl. Agarose gel electrophoresis indicated no visible band for the remaining circular DNA. 2-Fold molar
excess of a scaffold 46-mer was annealed to the linear single-stranded DNA at a concentration of 100 ng/ml by heating at 67 °C for 4 min
followed by slow cooling to room temperature over a period of 3-4 h.
The proportion of circularized vector as determined by agarose gel
electrophoresis was 35-45%. Fifty-fold molar excess of the modified
or unmodified hexanucleotide was ligated into the gap of this annealed
DNA in the presence of 20 units of T4 DNA ligase (Invitrogen) in 40 mM Tris-HCl buffer, pH 7.8, 8 mM MgCl2, 16 mM dithiothreitol, and 1 mM ATP at 16 °C overnight. A "mock ligation" was
also carried out in which no oligonucleotide was included.
SOS Induction and Transformation in E. coli--
E.
coli cells were grown in 100-ml cultures to 1 × 108 cells/ml and then harvested by centrifugation at
5,000 × g for 15 min at 0 °C. All E. coli cells
were grown in Luria broth, except the dnaQ49 strain NR11446,
which was grown in minimal medium. The cells were resuspended in an
equal volume of ice-cold deionized water and recentrifuged at 5000 × g for 30 min. This procedure was repeated except the
cells were resuspended in 50 ml of water. The bacterial pellet was
resuspended in 1 ml of glycerol/water (10% v/v) and kept on ice until
further use. To induce SOS, the following additional steps were
introduced after the first centrifugation. The cells were resuspended
in 50 ml of 10 mM MgSO4 and treated with UV
light (254 nm) (either 20 or 50 J/m2) in 25-ml aliquots in
150 × 50-mm plastic Petri dishes. The cultures were incubated in
Luria broth at 37 °C for 40 min in order to express SOS functions
maximally. Following SOS induction, these cells were centrifuged,
deionized, and resuspended in glycerol/water in a similar manner as
described earlier (13) except all manipulations were carried out in
subdued light.
Before transformation, the constructed genome was subjected to another
round of EcoRI treatment to digest any uncut or religated M13mp7L2 DNA. A 10-fold molar excess of a 46-mer that contained the DNA
sequence complementary to the scaffold oligomer was added to each DNA
solution, and the mixture was heated at 100 °C for 2 min to remove
the scaffold and rapidly cooled to 0 °C. To monitor the extent of
removal of the scaffold, a gapped genome was taken through the same
protocol. An aliquot of each of these DNA solutions was subjected to
agarose gel electrophoresis to ensure that the scaffold was
quantitatively denatured. For each transformation, 40 µl of the cell
suspension was mixed with 4 µl (500 ng) of DNA solution and
transferred to the bottom of an ice-cold Bio-Rad Gene-Pulser cuvette
(0.1-cm electrode gap). Electroporation of cells was carried out in a
Bio-Rad Gene-Pulser apparatus at 25 microfarads and 1.8 kV with the
pulse controller set at 200 ohms. Immediately after electroporation, 1 ml of SOC medium was added, and the mixture was transferred to a 1.5-ml
microcentrifuge tube. Part of the cells was plated following a 15-min
recovery at 37 °C in the presence of the plating bacteria E. coli GW5100, IPTG, and X-gal to determine the number of
independent transformants. The remainder of cells was centrifuged at
15000 × g for 5 min to isolate the phage-containing
supernatant. Minus-two and plus-one mutant phages were detected
directly from the progeny as blue plaques after 18 h of incubation
at 37 °C. All other colorless mutants were detected and isolated by
oligonucleotide hybridization as described (14).
Modeling Studies--
The molecular mechanics program DUPLEX
(21, 22) was employed to obtain energy minimized structures for the AP
and 1,8-ANP adducts in slipped mutagenic intermediate structures (23)
containing a two-base bulge. Optimized geometries for the AP and
1,8-ANP adducts were computed in modified nucleosides, using
Hartree-Fock calculations with 6-31 G* basis set, employing the
program package Gaussian 98 (24). These provided the bond lengths, bond
angles, and dihedral angles needed for the coordinate generator in
DUPLEX. Gaussian 98 (24) was also employed to compute the partial
charges for these adducts in modified nucleosides. CNDO calculations, which provide charges that are compatible with the rest of the DUPLEX
partial charge set (21), were used for this purpose. These are given in
Table S1 of the Supplemental Material. Other DUPLEX force field
parameters for the AP and 1,8-ANP adducts are the same as those
employed previously (25).
 |
RESULTS |
Construction and Characterization of M13 Genomes Containing a
Single Adduct--
Deoxyhexanucleotides, d(CGCG*CG) containing the C8
guanine adduct of 1-NP, 1,6-DNP, or 1,8-DNP, were synthesized by
reacting N-hydroxy derivative of 1-aminopyrene or
1-amino-6(8)-nitropyrene in
N,N-dimethylformamide/water (1:9), pH 5.0-5.5,
for 16-20 h at ambient temperature and purified by HPLC followed by
PAGE (19, 20). The adducted oligonucleotides were characterized by
enzymatic digestion to nucleosides followed by HPLC analysis and by
electrophoretic migration pattern of the fragments after piperidine
cleavage (at 90 °C for 1 h) in comparison with Maxam-Gilbert G
reaction products of the unmodified hexamer (see Refs. 13 and 20 for
details). They were also examined by electrospray ionization-mass
spectrometry analysis. The negative ion electrospray ionization-mass
spectra of the hexamer containing Gua-C8-AP, Gua-C8-1,6-ANP, and
Gua-C8-1,8-ANP contained an intense M
H peak at 2007.45 Da
(theoretical 2007.36), 2052.39 Da (theoretical 2052.34), and 2052.52 Da
(theoretical 2052.34), respectively. It is noteworthy that in each
synthesis the major product contained the C8 guanine adduct at the
second G, which eluted 7-8 min after the unmodified peak and 6-12 min before the other modified hexamers by reverse-phase HPLC (see Fig. S1
of the Supplemental Material for a typical chromatogram). Reinjection
of the purified d(CGCG*CG) on a reverse-phase column showed no
detectable level of either the unmodified or the other modified
hexamers, and based on the chromatographic analysis, we estimated that
the modified hexamers were in excess of 98% pure. Since further PAGE
purification of each oligonucleotide was carried out, and the migration
pattern of each adducted hexamer was different (data not shown), the
purity of the hexamers used in this work was expected to be
significantly higher than 98%. Fig. 2
shows a typical autoradiogram of the unmodified and the three adducted
hexamers by denaturing PAGE.

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Fig. 2.
Denaturing PAGE of
32P-end-labeled d(CGCGCG) (lane 1), and
d(CGCG1,8-ANPCG) (lane 2),
d(CGCGAPCG) (lane 3), and
d(CGCG1,6-ANPCG) (lane 4).
Electrophoresis was carried out at room temperature at 1800 V for
3.5 h on a 16% polyacrylamide gel (30 × 40 cm × 0.4 mm) containing 8 M urea.
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The hairpin region of a +2 frameshift mutant clone of M13mp7 was
digested with EcoRI (26). This was recircularized
noncovalently by annealing a scaffold 46-mer, the two ends of which
were complementary to the terminal 20 nucleotides of the linearized
vector. The central segment of the oligomer was complementary to
5'-CGCGCG, which allowed the ligation of 5'-CGCG*CG to the ends of M13
vector by T4 DNA ligase. A control (i.e. unmodified) genome
was constructed in a similar manner. A mock ligation was also carried
out, in which no oligonucleotide was added.
In order to visualize the unmodified and modified DNA constructs, a
portion of each of these genomes was run on a 1% agarose gel in the
presence of ethidium bromide. Densitometry analysis indicated that the
efficiency of recircularization of the unmodified and the three
modified vectors was 34-35%. When these DNA constructs were treated
with an excess of BssHII, >95% of the unmodified genome
was digested to a linear material, whereas the presence of the adduct
at the restriction site of the modified genome prevented it from being
digested with this enzyme. This is expected because inhibition of
restriction enzyme cleavage by the presence of DNA adducts including
Gua-C8-AP has been shown in many earlier studies (13).
To remove the 46-mer scaffold from the M13 DNA, each DNA solution was
heated at 100 °C for 2 min and rapidly cooled to 0 °C. Prior to
heating, a 10-fold molar excess of a 46-mer that contained the DNA
sequence complementary to the scaffold oligomer was added to the DNA
solution to ensure that the scaffold, once denatured, did not reanneal
on the M13 DNA. As shown in Fig. 3,
subsequent to removal of the scaffold 46-mer, a band for the circular
DNA construct was observed on an agarose gel when either a control or
adducted hexamer was included in the ligation mix, but no circular DNA
was detectable (<2%) in the mock construct.

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Fig. 3.
Characterization of the M13 constructs.
The DNA samples were electrophoresed on 1% agarose gel in the presence
of ethidium bromide (1 µg/ml). Lanes 1 and 9 show the single-stranded M13mp7L2 DNA. Lanes 2 and
3 show EcoRI-digested M13mp7L2 before and after
annealing the 46-mer scaffold, respectively. The M13 genomes after
ligation of unmodified and 1,6-ANP-dG-, 1,8-ANP-dG-, and AP-dG-modified
hexamers followed by removal of the scaffold 46-mer are shown in
lanes 4-7, respectively. Lane 8 is the same as
in lanes 4-7 except no hexamer was included in the ligation
mixture (i.e. mock).
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Mutagenesis of the Three Adducts in Repair-proficient and
-deficient Background--
The constructed M13 vector used in this
study is a +2 derivative, which should generate colorless plaques in
the presence of IPTG and X-gal. Either a
2 or a +1 frameshift can
restore the reading frame to a Lac+ phenotype generating
blue plaques. Spontaneous mutagenesis of the unmodified vector as
determined phenotypically by the reversion to blue plaques was 2 × 10
3 or less (Table I).
In the repair-competent strain DL7 the presence of Gua-C8-AP increased
the MF to 1.7% as determined by an increased proportion of blue
plaques (Table I and Fig. 4A).
With SOS (50 J/m2), the MF of Gua-C8-AP increased to 1.9%.
The MF of the two DNP adducts, Gua-C8-1,6-ANP and Gua-C8-1,8-ANP,
were 6.8 and 10.1% without SOS, which increased to 8.2 and 12.8%,
respectively, with SOS (20 J/m2). SOS induction with a
higher dosage of UV (50 J/m2) resulted in further increase
in MF of the two DNP adducts, which was found to be 11.2 and 15.0%,
respectively, for Gua-C8-1,6-ANP and Gua-C8-1,8-ANP (Table I and Fig.
4A). In another repair-competent strain, NR9295, the numbers
were somewhat lower, although the trend was similar (Table I and Fig.
4B).
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Table I
Mutagenesis detected phenotypically at the (CG)3 sequence
The numbers shown are the average of 2-6 experiments.
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Fig. 4.
Comparison of CpG deletion mutation frequency
of control, dG-C8-AP, dG-C8-1,6-ANP, and dG-C8-1,8-ANP containing M13
genome in repair-competent (DL7) and uvrA (DL6)
strains (A) and repair-competent (NR9295),
mutS (NR9294), and dnaQ (NR11446)
strains (B).
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In a nucleotide excision repair-impaired strain (uvrA) the
MF did not increase without SOS, but with SOS (20 J/m2)
there was a notable increase in MF, much more so than what we observed
for the repair-proficient strains (Table I and Fig. 4A).
Even though these bulky adducts are likely to be repaired by the
nucleotide excision repair system, in single-stranded DNA it is
unlikely to have a significant effect. We believe that the higher MF
with SOS was due to a more pronounced SOS response, which in turn was a
result of the persistence of the UV lesions in the E. coli
DNA. For the mismatch repair-deficient strain (mutS), there
was ~2-fold increase in MF of each of the three adducts compared with
the repair-proficient strain. In the dnaQ strain, the
increase in MF was most pronounced, and for Gua-C8-AP in the absence of
SOS, MF was ~8-fold of the same in repair-competent strain, which
increased to 11-fold with SOS (Table I and Fig. 4B). The
extent of increase in MF for the two DNP adducts was much less
pronounced in the dnaQ strain. Both with and without SOS, MF
was ~3-fold relative to the repair-competent strain. A population of
blue plaques from each transformation was subjected to DNA sequencing.
In each case more than 90% of the blue plaques contained CpG
deletions, although a small population of one-base additions was also detected.
To investigate phenotypically undetectable base substitutions and other
types of frameshifts, we used oligonucleotide hybridization with a
17-mer probe complementary to the region of M13 where the 6-mer was
inserted. The probe was designed to bind only to the non-mutant
plaques. Therefore, all non-hybridized or weakly hybridized plaques
were considered putative mutants and subjected to DNA sequencing. In
the progeny from the control construct with SOS ~1% progeny
(13/1357) were mutants, which showed one-base deletions in various
sites in the CGCGCG insert. The MF of clear plaques did not increase in
the adducted vectors, although both base substitutions and one-base
deletions were detected. We conclude that in this repetitive CpG
sequence, the predominant mutation induced by the three adducts is
2 frameshifts.
Modeling Studies--
To investigate possible structural reasons
for the enhancement in two-base deletions when the adducts contained a
nitro group on the pyrene ring, we have carried out a computer modeling
study. Specifically, we wished to obtain structures of the slipped
mutagenic intermediate, which has been proposed to cause two-base
frameshifts in NarI-type sequences (23). We investigated the
following Sequence 1,
with G4* modified by AP or 1,8-ANP. An extensive conformational
search for slipped mutagenic intermediate structures of this type with
modification at guanine C8 by another aromatic amine, N-acetyl-2-aminofluorene (AAF), provided a reasonable
starting model (27). AAF was replaced by AP or 1,8-ANP; the base
sequence was adjusted to match that in the present work (C1 replacing a G), and the structure was energy-minimized. A recently improved version
of the molecular mechanics program DUPLEX (21), which accurately
reproduces detailed sequence-dependent conformational features of B-DNA duplexes (22), was employed in this work. In this
improved DUPLEX, solvent water is treated implicitly with a new steep,
sigmoidal distance-dependent dielectric function that
levels off to the dielectric constant of water at an interatomic distance equivalent to about one solvation shell. High level quantum mechanical calculations were used to obtain geometries of the AP and
1,8-ANP. These revealed that the oxygens of the NO2 are out
of plane with the AP ring system, with a dihedral angle
O1-N-C9-C10 (see Fig. 1) of
31.6 degrees. An in-plane
position of these oxygens produces steric crowding with hydrogens in
the adjacent ring system. The DUPLEX molecular mechanics calculations
employed this quantum mechanically derived orientation of the
NO2.
Stereo views of the modeled structures are shown in Fig.
5. In both cases, a base-displaced
intercalated conformation is adopted with the aromatic ring system
stacked on the G6-C9 base pair. The modified, unpartnered G4* has a
syn-glycosidic torsion angle and is displaced into the major
groove, with the covalently linked pyrene ring system directed toward
the minor groove; the bulged out, unpartnered C5 is positioned on the
minor groove side and interacts on one face with the edge of the pyrene
ring system. These structures share features with the NMR solution
structure of the AP adduct in a DNA duplex with normal partner C in
adopting a syn-guanine base-displaced intercalated
conformation, with the modified G displaced into the major groove (28).
In the duplex structure the looped out partner C was displaced into the
major groove, and the NMR data indicated conformational heterogeneity for this residue. Key torsion angles
,
' and
' defining the orientation of the carcinogenic moiety (
, O4'-C1'-N9-C4;
', N9-C8-N(AP)-C1(AP);
', C8-N(AP)-C1(AP)-C10A(AP)) (Fig. 1) are very similar in our modeled slippage structures and in the NMR solution
structure of the duplex (see Table S2 of the Supplemental Material).

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Fig. 5.
Stereo views of DUPLEX generated 2
frameshift structures of the AP adduct and 1,8-ANP adduct.
A, the AP adduct. B, the 1,8-ANP adduct.
C, the 1,8-ANP adduct and the proposed water-mediated
hydrogen bond network. The AP and 1,8-ANP are in green. The
adducted G4 is in magenta, and the bulged out C5 is in
cyan. The atoms involved in the hydrogen bond network are
colored by atom types. The N of the nitro group is blue and
the two oxygens are red. Also designated in red
are two other key oxygens: O2 of C5 and O4' of the G6 sugar. The water
oxygens are red, and hydrogens are white. Other
residues and atoms are in gray.
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Even though the slippage structures for the AP and 1,8-ANP are very
similar, a key feature distinguishes them that can account for greater
stability in the case of the 8-nitro derivative. Specifically, the
nitro group oxygens are placed in a position to permit water-mediated hydrogen bonds between the carbonyl oxygen of the looped out C5 and one
of the nitro oxygens; furthermore, this same nitro-oxygen can also form
a second water-mediated hydrogen bond to the sugar O4' of G6. Table S3
of the Supplemental Material gives geometric features of these hydrogen
bonds. In Fig. 5C we have modeled in these waters
explicitly. This network of water-mediated hydrogen bonds is a feature
only of the nitro-containing Gua-C8-1,8-ANP and would serve to
stabilize its slippage structure, including especially the looped out
C; the latter would be more mobile in the AP slippage structure, as is
the looped out partner C to the AP-modified G in the NMR solution
structure of the duplex.
 |
DISCUSSION |
This study unequivocally demonstrated that the guanine C8 adducts
of 1,6- and 1,8-DNP are significantly more mutagenic in E. coli cells than the 1-NP adduct. With SOS, MF of each adduct increases. The trend is similar in both repair-competent and
repair-impaired cells, even though the magnitude of frameshift is
highly dependent on the type of repair defect.
We hypothesize that the mechanism of two-base deletion involves
generation of a promutagenic slipped frameshift intermediate whose
structure is similar in the different adducts, but whose stability is
greater in the more mutagenic adduct. Whereas these intermediates may
form spontaneously in repetitive sequences (29), certain lesions such
as the guanine C8 adducts of nitropyrenes and AAF are particularly
efficient in generating such intermediates in high frequency (13, 30).
Scheme I shows our working model and also
suggests how the slippage and elongation might be linked to
proofreading and mismatch repair activity. To investigate if this may
be the case, we have performed a computer modeling study comparing
Gua-C8-AP with Gua-C8-1,8-ANP. This study supports our hypothesis and
shows how the 8-nitro substituent can stabilize the looped structure in
solution.

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|
Scheme I.
A model to account for the CpG deletion
mutagenesis in the (CG)3 sequence and the effects of
proofreading and mismatch repair.
|
|
High fidelity of DNA replication is maintained by at least three major
steps (16). A high level of efficiency of the DNA polymerase in
incorporating correct nucleotides is certainly very important.
Nevertheless errors do occur, and in a large fraction of such cases the
3'-5'-exonuclease activity associated with the DNA polymerase can
remove the incorrectly incorporated nucleotides. In addition, a DNA
mismatch repair system detects and corrects mismatched nucleotides
shortly after replication. It has been estimated that nucleotide
selection discriminates against errors by 200,000-2,000,000-fold,
proofreading by 40-200-fold, and mismatch repair by 20-400-fold, each
depending on the type of error (31). However, the ability of the DNA
polymerase to incorporate the correct nucleotide opposite many
carcinogen-DNA adducts is impaired, and the types and frequencies of
misincorporation are often dependent on the DNA sequence surrounding
the lesion. It has been suggested that when the replicative polymerase,
pol III, encounters a replication-blocking lesion, it detaches from the
replication terminus and translesional polymerases are recruited (32).
Other polymerases such as pol II, pol IV, and pol V have been reported
to be involved at this stage for translesion synthesis (32-35). Some
of these bypass polymerases are error-prone. The current work indicates
the roles of two complementary repair systems in correcting the
promutagenic slipped frameshift intermediate. As we have suggested in
Scheme I, when either repair is impaired, there would be an increase in
CpG deletion events. The guanine C8 adduct formed by AAF, a potent
frameshift mutagenic lesion, which also intercalates with base
displacement (36) and promotes slipped frameshift intermediates, shares
some of the properties of the nitropyrene-DNA adducts (30, 34, 35, 37,
38). Our modeling studies of this slipped frameshift intermediate suggest that the bulged structure is more stable in the case of the
1,8-ANP compared with the AP adduct by virtue of its added hydrogen-bonding potential.
Current investigations in E. coli indicate that mutagenic
bypass of bulky lesions can be carried out by pol II, pol III, pol IV,
or pol V depending on the specific lesion and the base sequence context
(35, 39). Relaxed steric constraints in the active site appear to be a
feature common to Y family bypass DNA polymerases, such as pol IV (40,
41). Our modeling efforts suggest that specific hydrogen bonding
capabilities of the nitro group, through water-mediated hydrogen bonds
within the bulge, are plausible structural features that would
stabilize the slipped mutagenic intermediate more in the 1,8-ANP adduct
compared with the AP adduct itself. It is also conceivable that there
may exist stabilizing water-mediated or direct hydrogen-bonding
interactions of the nitro group with specific amino acid residues
within the polymerase. Since the 1,6-ANP adduct would also allow for
such unique hydrogen-bonding interactions, stabilization of a slipped
intermediate through an analogous structural mechanism could be envisioned.
Recent studies (32-35), including the current work, suggest
three major issues relating to translesion synthesis. First, it is
important to analyze the structure, conformation, and stability of the
promutagenic intermediate induced by the lesion, such as the slipped
frameshift intermediate described here. Second, the DNA polymerases
that can bypass the damage either with or without help from accessory
proteins play crucial roles in both error-free and error-prone bypass
events. This area of research has lately generated some intriguing data
(32, 42). Finally, the roles of DNA repair proteins that are involved
either before or after the replication process must also be taken into
account. It is fascinating how the different branches of chemistry and
genetics are merging to decipher these puzzles of replication and mutagenesis.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Roel Schaaper for the
dnaQ and mutS strains. The Oak Ridge National Laboratory is
managed by the University of Tennessee, Battelle, LLC, for the
United States Department of Energy under Contract
DE-AC05-00OR22725.
 |
FOOTNOTES |
*
This work was supported in part by NIEHS Grant ES09127 (to
A. K. B.) and NCI Grants CA75449 and CA28038 (to S. B.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Fig. S1 and Tables
S1-S3.
**
Recipient of Research Career Development Award 1 K02 ES00318 from
the NIEHS, National Institutes of Health. To whom correspondence should
be addressed. Tel.: 860-486-3965; Fax: 860-486-2981; E-mail: ashis.basu@uconn.edu.
Published, JBC Papers in Press, September 17, 2002, DOI 10.1074/jbc.M208103200
 |
ABBREVIATIONS |
The abbreviations used are:
1-NP, 1-nitropyrene;
AP, 1-aminopyrene;
Gua-C8-AP, the corresponding base,
N-(guanin-8-yl)-1-aminopyrene;
DNP, dinitropyrene;
1, 6-ANP, 1-amino-6-nitropyrene;
Gua-C8-1, 6-ANP,
N-(guanin-8-yl)-1-amino-6-nitropyrene;
1, 8-ANP,
1-amino-8-nitropyrene;
Gua-C8-1, 8-ANP,
N-(guanin-8-yl)-1-amino-8-nitropyrene;
AAF, N-acetyl-2-aminofluorene;
IPTG, isopropyl
-D-thiogalactopyranoside;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
Gua, guanine;
MF, mutation frequency;
HPLC, high pressure liquid
chromatography.
 |
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