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Structure of the 1,4-Bis(2‘-deoxyadenosin-<i>N</i><sup>6</sup>-yl)-2<i>S</i>,3<i>S</i>-butanediol Intrastrand DNA Cross-Link Arising from Butadiene Diepoxide in the Human N-<i>ras</i> Codon 61 Sequence
No full textThe 1,4-bis(2‘-deoxyadenosin-N6-yl)-2S,3S-butanediol intrastrand DNA cross-link arises from the bis-alkylation of tandem N6-dA sites in DNA by R,R-butadiene diepoxide (BDO2). The oligodeoxynucleotide
5‘-d(C1G2G3A4C5X6Y7G8A9A10G11)-3‘·5‘-d(C12T13T14C15T16T17G18T19C20C21G22)-3‘ contains the BDO2
cross-link between the second and third adenines of the codon 61 sequence (underlined) of the human
N-ras protooncogene and is named the (S,S)-BD-(61-2,3) cross-link (X,Y = cross-linked adenines). NMR
analysis reveals that the cross-link is oriented in the major groove of duplex DNA. Watson−Crick base
pairing is perturbed at base pair X6·T17, whereas base pairing is intact at base pair Y7·T16. The cross-link
appears to exist in two conformations, in rapid exchange on the NMR time scale. In the first conformation,
the β-OH is predicted to form a hydrogen bond with T16 O4, whereas in the second, the β-OH is predicted
to form a hydrogen bond with T17 O4. In contrast to the (R,R)-BD-(61-2,3) cross-link in the same sequence
(Merritt, W. K., Nechev, L. V., Scholdberg, T. A., Dean, S. M., Kiehna, S. E., Chang, J. C., Harris, T.
M., Harris, C. M., Lloyd, R. S., and Stone, M. P. (2005) Biochemistry 44, 10081−10092), the anti-conformation of the two hydroxyl groups at Cβ and Cγ with respect to the Cβ−Cγ bond results in a
decreased twist between base pairs X6·T17 and Y7·T16, and an approximate 10° bending of the duplex.
These conformational differences may account for the differential mutagenicity of the (S,S)- and (R,R)-BD-(61-2,3) cross-links and suggest that stereochemistry plays a role in modulating biological responses
to these cross-links (Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd,
R. S. (2002) Chem. Res. Toxicol. 15, 1572−1580)
Role of a Polycyclic Aromatic Hydrocarbon Bay Region Ring in Modulating DNA Adduct Structure: The Non-Bay Region (8<i>S</i>,9<i>R</i>,10<i>S</i>,11<i>R</i>)-<i>N</i><sup>6</sup>-[11-(8,9,10,11-Tetrahydro-8,9,10-trihydroxybenz[<i>a</i>]anthracenyl)]-2‘-deoxyadenosyl Adduct in Codon 61 of the Human <i>N-ras</i> Protooncogene<sup>†</sup>
No full textThe structure of the non-bay region (8S,9R,10S,11R)-N6-[11-(8,9,10,11-tetrahydro-8,9,10-trihydroxybenz[a]anthracenyl)]-2‘-deoxyadenosyl adduct at X6 of 5‘-d(CGGACXAGAAG)-3‘·5‘-d(CTTCTTGTCCG)-3‘, incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene,
was determined. Molecular dynamics simulations were restrained by 475 NOEs from 1H NMR. The benz[a]anthracene moiety intercalated above the 5‘-face of the modified base pair and from the major groove.
The duplex suffered distortion at and immediately adjacent to the adduct site. This was evidenced by the
disruption of the Watson−Crick base pairing for X6·T17 and A7·T16 and the increased rise of 7.7 Å between
base pairs C5·G18 and X6·T17. Increased disorder was observed as excess line width of proton resonances
near the lesion site. Comparison with the bay region benzo[a]pyrene [Zegar, I. S., Kim, S. J., Johansen,
T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212−6224]
and bay region benz[a]anthracene [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T.
M., and Stone, M. P. (1999) Biochemistry 38, 2969−2981] adducts with the corresponding stereochemistry
and at the same site shows that this non-bay region benz[a]anthracene lesion assumes different base pair
geometry, in addition to exhibiting greater disorder. These differences are attributed to the loss of the bay
region ring. The results suggest the bay region ring contributes to base stacking interactions at the lesion
site. These structural differences between the non-bay and bay region lesions are correlated with site-specific mutagenesis data. The bay region benzo[a]pyrene and bay region benz[a]anthracene adducts were
poorly replicated in vivo, and induced A → G mutations. In contrast, the non-bay region benz[a]anthracene
adduct was easily bypassed in vivo and was nonmutagenic
Structure of the 1,4-Bis(2‘-deoxyadenosin-<i>N</i><sup>6</sup>-yl)-2<i>S</i>,3<i>S</i>-butanediol Intrastrand DNA Cross-Link Arising from Butadiene Diepoxide in the Human N-<i>ras</i> Codon 61 Sequence
No full textThe 1,4-bis(2‘-deoxyadenosin-N6-yl)-2S,3S-butanediol intrastrand DNA cross-link arises from the bis-alkylation of tandem N6-dA sites in DNA by R,R-butadiene diepoxide (BDO2). The oligodeoxynucleotide
5‘-d(C1G2G3A4C5X6Y7G8A9A10G11)-3‘·5‘-d(C12T13T14C15T16T17G18T19C20C21G22)-3‘ contains the BDO2
cross-link between the second and third adenines of the codon 61 sequence (underlined) of the human
N-ras protooncogene and is named the (S,S)-BD-(61-2,3) cross-link (X,Y = cross-linked adenines). NMR
analysis reveals that the cross-link is oriented in the major groove of duplex DNA. Watson−Crick base
pairing is perturbed at base pair X6·T17, whereas base pairing is intact at base pair Y7·T16. The cross-link
appears to exist in two conformations, in rapid exchange on the NMR time scale. In the first conformation,
the β-OH is predicted to form a hydrogen bond with T16 O4, whereas in the second, the β-OH is predicted
to form a hydrogen bond with T17 O4. In contrast to the (R,R)-BD-(61-2,3) cross-link in the same sequence
(Merritt, W. K., Nechev, L. V., Scholdberg, T. A., Dean, S. M., Kiehna, S. E., Chang, J. C., Harris, T.
M., Harris, C. M., Lloyd, R. S., and Stone, M. P. (2005) Biochemistry 44, 10081−10092), the anti-conformation of the two hydroxyl groups at Cβ and Cγ with respect to the Cβ−Cγ bond results in a
decreased twist between base pairs X6·T17 and Y7·T16, and an approximate 10° bending of the duplex.
These conformational differences may account for the differential mutagenicity of the (S,S)- and (R,R)-BD-(61-2,3) cross-links and suggest that stereochemistry plays a role in modulating biological responses
to these cross-links (Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd,
R. S. (2002) Chem. Res. Toxicol. 15, 1572−1580)
Intercalation of the (1<i>R</i>,2<i>S</i>,3<i>R</i>,4<i>S</i>)-<i>N</i><sup>6</sup>-[1-(1,2,3,4- Tetrahydro-2,3,4-trihydroxybenz[<i>a</i>]anthracenyl)]-2‘-deoxyadenosyl Adduct in the N-<i>ras</i> Codon 61 Sequence: DNA Sequence Effects<sup>†</sup>
No full textThe structure of the bay region (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2‘-deoxyadenosyl adduct at X7 of 5‘-d(CGGACAXGAAG)-3‘·5‘-d(CTTCTTGTCCG)-3‘, incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined
by NMR. This was the bay region benz[a]anthracene RSRS (61,3) adduct. The BA moiety intercalated
above the 5‘-face of the modified base pair. NOE connectivities between imino protons were disrupted at
T16 and T17. Large chemical shifts at the lesion site were consistent with ring current shielding arising
from the BA moiety. A large chemical shift dispersion was observed for the BA aromatic protons. An
increased rise of 8.17 Å was observed between base pairs A6·T17 and X7·T16. The PAH moiety stacked
with the purine ring of A6, the 5‘-neighbor nucleotide. This resulted in buckling of the 5‘-neighbor A6·T17
base pair, evidenced by exchange broadening for the T17 imino resonance. It also interrupted sequential
NOE connectivities between nucleotides C5 and A6. The A6 deoxyribose ring showed an increased
percentage of the C3‘-endo conformation. This differed from the bay region BA RSRS (61,2) adduct, in
which the lesion was located at position X6 [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M.,
Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969−2981], but was similar to the benzo[a]pyrene BP SRSR (61,3) adduct [Zegar I. S., Chary, P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd,
R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Biochemistry 37, 16516−16528]. The altered
sugar pseudorotation at A6 appears to be common to both bay region BA RSRS (61,3) and BP SRSR
(61,3) adducts. It could not be discerned if the C3‘-endo conformation at A6 in the BA RSRS (61,3)
adduct altered base pairing geometry at X7·T16, as compared to the C2‘-endo conformation. The structural
studies suggest that the mutational spectrum of this adduct may be more complex than that of the BA
RSRS (61,2) adduct
Stereospecific Structural Perturbations Arising from Adenine N<sup>6</sup> Butadiene Triol Adducts in Duplex DNA
No full textButadiene is oxidized in vivo to form stereoisomeric butadiene diol epoxides (BDE). These
react with adenine N6 in DNA yielding stereoisomeric N6-(2,3,4-trihydroxybutyl)-2‘-deoxyadenosyl (BDT) adducts. When replicated in Escherichia coli, the (2R,3R)-N6-(2,3,4-trihydroxybutyl)-2‘-deoxyadenosyl adduct yielded low levels of A→G mutations whereas the (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2‘-deoxyadenosyl butadiene triol adduct yielded low levels of A→C
mutations [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000)
Environ. Mol. Mutagen. 35, 48−56]. Accordingly, the structure of the (2R,3R)-N6-(2,3,4-trihydroxybutyl)-2‘-deoxyadenosyl adduct at position X6 in d(CGGACXAGAAG)·d(CTTCTTGTCCG),
the ras61 R,R-BDT-(61,2) adduct, was compared to the corresponding structure for the (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2‘-deoxyadenosyl adduct in the same sequence, the ras61 S,S-BDT-(61,2) adduct. Both the R,R-BDT-(61,2) and S,S-BDT-(61,2) adducts are oriented in the major
groove of the DNA, accompanied by modest structural perturbations. However, structural
refinement of the two adducts using a simulated annealing restrained molecular dynamics
(rMD) approach suggests stereospecific differences in hydrogen bonding between the hydroxyl
groups located at the β- and γ-carbons of the BDT moiety, and T17 O4 of the modified base pair
X6·T17. The rMD calculations predict hydrogen bond formation between the γ-OH and the T17
O4 in the R,R-BDT-(61,2) adduct whereas in the S,S-BDT-(61,2) adduct, hydrogen bond
formation is predicted between the β-OH and the T17 O4. This difference positions the two
adducts differently in the major groove. This may account for the differential mutagenicity of
the two adducts and suggests that the two adducts may interact differentially with other DNA
processing enzymes. With respect to mutagenesis in E. coli, the minimal perturbation of DNA
induced by both major groove adducts correlates with their facile bypass by three E. coli DNA
polymerases in vitro and may account for their weak mutagenicity [Carmical, J. R., Nechev,
L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48−56]
Major Groove (<i>S</i>)-α-(<i>N</i><sup>6</sup>-Adenyl)styrene Oxide Adducts in an Oligodeoxynucleotide Containing the Human <i>N-ras</i> Codon 61 Sequence: Conformations of the S(61,2) and S(61,3) Sequence Isomers from <sup>1</sup>H NMR<sup>†</sup>
No full textThe
(S)-α-(N6-adenyl)styrene oxide
adducts at positions X6 in
d(CGGACXAGAAG)·
d(CTTCTTGTCCG) and X7 in
d(CGGACAXGAAG)·d(CTTCTTGTCCG), incorporating codons 60,
61
(underlined), and 62 of the human n-ras protooncogene, were
examined by 1H NMR. These were the
S(61,2) and S(61,3) adducts. Chemical shift perturbations were in
the 3‘-direction from the sites of
adduction; upfield changes associated with the styrene aromatic ring
current were noted for S-SOA6 H2
and H1‘, T16 N3H, H6, and CH3 resonances in the
S(61,2) oligomer. In the S(61,3) oligomer,
S-SOA7 H1‘,
T16 H1‘, C15 N4Ha, and H5 shifted
upfield. The styrene aromatic rings flipped rapidly on the NMR
time
scale; under these conditions the ortho and meta aromatic protons were
equivalent. A sequence effect, in
which the S(61,2) adduct equilibrated between two conformers, while the
S(61,3) adduct exhibited only
a single conformation, was observed. Potential energy minimization
of the S(61,2) adduct major
conformation yielded a structure in which the styrene ring was oriented
in the 3‘-direction and interacted
primarily with the complementary strand. For the S(61,3) adduct,
291 restraints were obtained from
NOE data at three mixing times using relaxation matrix analysis.
The emergent structures refined to an
average rms difference of 1.3 Å, determined by pairwise analysis.
These were compared to NOE intensity
data; the calculated sixth root residual index was 9.2 ×
10-2 at 250 ms. In the refined structure,
the
styrene ring was also oriented in the 3‘-direction and interacted with
the complementary strand. The
minor conformation of the S(61,2) adduct was not identified. These
results contrasted with the
diastereomeric R(61,2) and R(61,3) adducts, which underwent slow ring
flips on the NMR time scale and
for which small sequence effects involving the minimium energy
conformation of the styrene ring were
observed [Feng, B., Zhou, L., Passarelli, M., Harris, C. M., Harris,
T. M., & Stone, M. P. (1995)
Biochemistry 34, 14021−14036]
Structure of an Oligodeoxynucleotide Containing a Butadiene Oxide-Derived N1 Beta-Hydroxyalkyl Deoxyinosine Adduct in the Human <i>N-ras</i> Codon 61 Sequence<sup>†</sup>
No full textThe solution structure of the N1-(1-hydroxy-3-buten-2(S)-yl)-2‘-deoxyinosine adduct arising
from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine,
was determined, in the oligodeoxynucleotide d(CGGACXAGAAG)·d(CTTCTCGTCCG). This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene,
and was named the ras61 S-N1−BDO-(61,2) adduct. 1H NMR revealed a weak C5 H1‘ to X6 H8 NOE,
followed by an intense X6 H8 to X6 H1‘ NOE. Simultaneously, the X6 H8 to X6 H3‘ NOE was weak. The
resonance arising from the T17 imino proton was not observed. 1H NOEs between the butadiene moiety
and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 364
NOE-derived distance restraints yielded a structure in which the modified deoxyinosine was in the high
syn conformation about the glycosyl bond, and T17, the complementary nucleotide, was stacked into the
helix, but not hydrogen bonded with the adducted inosine. The refined structure provided a plausible
hypothesis as to why this N1 deoxyinosine adduct strongly coded for the incorporation of dCTP during
trans lesion DNA replication, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J.,
Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292−296], and in
mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R.
S. (2002) Chem. Res. Toxicol. 15, 1572−1580]. Rotation of the N1 deoxyinosine adduct into the high syn
conformation may facilitate incorporation of dCTP via Hoogsteen-type templating with deoxyinosine,
thus generating A-to-G mutations
Intercalation of the (1<i>S</i>,2<i>R</i>,3<i>S</i>,4<i>R</i>)-<i>N</i><sup>6</sup>- [1-(1,2,3,4-Tetrahydro-2,3,4-trihydroxybenz[<i>a</i>]anthracenyl)]-2‘-deoxyadenosyl Adduct in an Oligodeoxynucleotide Containing the Human <i>N</i>-<i>ras</i> Codon 61 Sequence<sup>†</sup>
No full textThe (1S,2R,3S,4R)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2‘-deoxyadenosyl adduct at X6 of 5‘-d(CGGACXAGAAG)-3‘·5‘-d(CTTCTTGTCCG)-3‘, incorporating codons 60,
61 (underlined), and 62 of the human N-ras protooncogene, results from trans opening of (1R,2S,3S,4R)-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Two conformations
of this adduct exist, in slow exchange on the NMR time scale. A structure for the major conformation,
which represents approximately 80% of the population, is presented. In this conformation, an anti glycosidic
torsion angle is observed for all nucleotides, including S,R,S,RA6. The refined structure is a right-handed
duplex, with the benz[a]anthracene moiety intercalated on the 3‘-face of the modified base pair, from the
major groove. It is located between S,R,S,RA6·T17 and A7·T16. Intercalation is on the opposite face of the
modified S,R,S,RA6·T17 base pair as compared to the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2‘-deoxyadenosyl adduct, which intercalated 5‘ to the modified R,S,R,SA6·T17 base pair [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P.
(1999) Biochemistry 38, 2969−2981]. The spectroscopic data do not allow refinement of the minor
conformation, but suggest that the adenyl moiety in the modified nucleotide S,R,S,RA6 adopts a syn glycosidic
torsion angle. Thus, the minor conformation may create greater distortion of the DNA duplex. The results
are discussed in the context of site-specific mutagenesis studies which reveal that the S,R,S,RA6 lesion is
less mutagenic than the R,S,R,SA6 lesion
Unraveling the Aflatoxin−FAPY Conundrum: Structural Basis for Differential Replicative Processing of Isomeric Forms of the Formamidopyrimidine-Type DNA Adduct of Aflatoxin B<sub>1</sub>
No full textAflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes
base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists
as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable
but interconvertible forms that have been assigned by various workers as functional, positional, or
conformational isomers. Recently, this structural question became important when one of the two major
FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M.
E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 6655−6660]. NMR studies carried out on the AFB−FAPY bases and deoxynucleoside
3‘,5‘-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5
formyl derivatives involving axial asymmetry around the congested pyrimidine C5−N5 bond. Anomerization
of the protected β-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization
and furanosyl → pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY
species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the
mutagenic species. It has previously been assigned as the β anomer by NMR (Mao, H.; Deng, Z. W.;
Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374−4387). In single-stranded environments
the dominant species is the α anomer; it is a block to replication
Structure of a Site Specific Major Groove (2<i>S</i>,3<i>S</i>)-N<sup>6</sup>-(2,3,4-Trihydroxybutyl)-2‘-deoxyadenosyl DNA Adduct of Butadiene Diol Epoxide
No full textThe solution structure of the (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2‘-deoxyadenosyl adduct arising
from the alkylation of adenine N6 at position X6 in d(CGGACXAGAAG)·d(CTTCTTGTCCG), by
butadiene diol epoxide, was determined. This oligodeoxynucleotide contains codon 61 (underlined)
of the human N-ras protooncogene. This oligodeoxynucleotide, containing the adenine N6 adduct
butadiene triol (BDT) adduct at the second position of codon 61, was named the ras61 S,S-BDT-(61,2) adduct. NMR spectroscopy revealed modest structural perturbations localized to the site of
adduction at X6·T17, and its nearest-neighbor base pairs C5·G18 and A7·T16. All sequential NOE
connectivities arising from DNA protons were observed. Torsion angle analysis from COSY data
suggested that the deoxyribose sugar at X6 remained in the C2‘-endo conformation. Molecular
dynamics calculations using a simulated annealing protocol restrained by a total of 442 NOE-derived distances and J coupling-derived torsion angles refined structures in which the BDT moiety
oriented in the major groove. Relaxation matrix analysis suggested hydrogen bonding between the
hydroxyl group located at the β-carbon of the BDT moiety and the T17 O4 of the modified base pair
X6·T17. The minimal perturbation of DNA induced by this major groove adduct correlated with its
facile bypass by three Escherichia coli DNA polymerases in vitro and its weak mutagenicity
[Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol.
Mutagen. 35, 48−56]. Overall, the structure of this adduct is consistent with an emerging pattern
in which major groove adenine N6 alkylation products of styrene and butadiene oxides that do not
strongly perturb DNA structure are not strongly mutagenic
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