21 research outputs found
DNA base sequence effects on bulky lesion-induced conformational heterogeneity during DNA replication
4-Aminobiphenyl (ABP) and its structure analog 2-aminofluorene (AF) are well-known carcinogens. In the present work, an unusual sequence effect in the 5′-CTTCTG1G2TCCTCATTC-3′ DNA duplex is reported for ABP- and AF-modified G. Specifically, the ABP modification at G1 resulted in a mixture of 67% major groove B-type (B) and 33% stacked (S) conformers, while at the ABP modification at G2 exclusively resulted in the B-conformer. The AF modification at G1 and G2 lead to 25%:75% and 83%:17% B:S population ratios, respectively. These differences in preferred conformation are due to an interplay between stabilizing (hydrogen bonding and stacking that is enhanced by lesion planarity) and destabilizing (solvent exposure) forces at the lesion site. Furthermore, while the B-conformer is a thermodynamic stabilizer and the S-conformer is a destabilizer in duplex settings, the situation is reversed at the single strands/double strands (ss/ds) junction. Specifically, the twisted biphenyl is a better stacker at the ss/ds junction than the coplanar AF. Therefore, the ABP modification leads to a stronger strand binding affinity of the ss/ds junction than the AF modification. Overall, the current work provides conformational insights into the role of sequence and lesion effects in modulating DNA replication
Conformational and Thermodynamic Properties Modulate the Nucleotide Excision Repair of 2-Aminofluorene and 2-Acetylaminofluorene dG Adducts in the NarI Sequence
Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G1, G2 or G3 of NarI sequence (5′-CCG1G2CG3CC-3′). Our 19F-NMR/ICD results showed that FAAF at G1 and G3 prefer syn S-and W-conformers, whereas anti B-conformer was predominant for G2. We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G3 and-G1 duplexes incised more efficiently than the B-type G2 duplex (G3∼G1\u3eG2). The melting and thermodynamic data indicate that the S-and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2-to 3-fold less than FAAF: however, the incision efficiency was in order of G2∼G1\u3eG 3, a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts
Unusual Sequence Effects on Nucleotide Excision Repair of Arylamine Lesions: DNA Bending/Distortion as a Primary Recognition Factor
The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a -G*C N- sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of ‘stacked’ (S) and ‘B-type’ (B) conformers, whereas the N-acetylated FAAF also samples a ‘wedge’ (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A- to -T polarity swap in the G*C A/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2- to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3\u27-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli
Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence
Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G1, G2 or G3 of NarI sequence (5′-CCG1G2CG3CC-3′). Our 19F-NMR/ICD results showed that FAAF at G1 and G3 prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G2. We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G3 and -G1 duplexes incised more efficiently than the B-type G2 duplex (G3∼G1 > G2). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G2∼G1 > G3, a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts
Structures of 2-acetylaminofluorene modified DNA revisited: Insight into conformational heterogeneity
Despite the extensive data on dG-AAF, the major DNA adduct derived from the model carcinogen 2-acetylaminofluorene, little is known with respect to its solution structures. Here, we provide NMR/CD evidence for three conformers of dG-AAF in duplex DNA: major groove B-type (B), base-displaced stacked (S), and minor groove wedge (W). The S/B/W-conformational heterogeneities were found to be sensitive to the nature of the flanking DNA sequence contexts and pH. © 2010 American Chemical Society
Conformational insights into the lesion and sequence effects for arylamine-induced translesion DNA synthesis: \u3csup\u3e19\u3c/sup\u3eF NMR, surface plasmon resonance, and primer kinetic studies
Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3′-next flanking base (GCT vs GCA; G, FABP, N-(2′-deoxyguanosin-8- yl)-4′-fluoro-4-aminobiphenyl; FAF, N-(2′-deoxyguanosin-8-yl)-7- fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair (Jain, V., Hilton, B., Lin, B., Patnaik, S., Liang, F., Darian, E., Zou, Y., Mackerell, A. D., Jr., and Cho, B. P. (2013) Nucleic Acids Res., 41, 869-880). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo-) and Dpo4. We obtained dynamic 19F NMR spectra for two 19-mer modified templates during primer elongation: GCA [d(5′-CTTACCATCGCAACCATTC-3′)] and GCT [d(5′-CTTACCATCGCTACCATTC-3′)]. We found that lesion stacking is favored in the GCT sequence compared to the GCA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP \u3e FAF and GCA \u3e GCT. Primer extension was stalled at (n) and near (n - 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct\u27s chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo- vs Dpo4). Steady-state kinetics analysis with Kf-exo- revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis. © 2014 American Chemical Society
General Reagent Free Route to pH Responsive Polyacryloyl Hydrazide Capped Metal Nanogels for Synergistic Anticancer Therapeutics
Herewith,
we report a facile synthesis of pH responsive polyacryloyl hydrazide
(PAH) capped silver (Ag) or gold (Au) nanogels for anticancer therapeutic
applications. A cost-effective instant synthesis of PAH-Ag or PAH-Au
nanoparticles (NPs) possessing controllable particle diameter and
narrow size distribution was accomplished by adding AgNO<sub>3</sub> or AuCl to the aqueous solution of PAH under ambient conditions
without using any additional reagent. PAH possessing carbonyl hydrazide
pendant functionality served as both reducing and capping agent to
produce and stabilize the NPs. The stability analysis by UV–vis,
dynamic light scattering, and transmission electron microscopy techniques
suggested that these NPs may be stored in a refrigerator for at least
up to 2 weeks with negligible change in conformation. The average
hydrodynamic size of PAH-Ag NPs synthesized using 0.2 mmol/L AgNO<sub>3</sub> changed from 122 to 226 nm on changing the pH of the medium
from 5.4 to 7.4, which is a characteristic property of pH responsive
nanogel. Camptothecin (CPT) with adequate loading efficiency (6.3%)
was encapsulated in the PAH-Ag nanogels. Under pH 5.4 conditions,
these nanogels released 78% of the originally loaded CPT over a period
of 70 h. The antiproliferative potential of PAH-Ag-CPT nanogels (at
[CPT] = 0.6 μg/mL) against MCF-7 breast adeno-carcinoma cells
were ∼350% higher compared to that of the free CPT as evidenced
by high cellular internalization of these nanogels. Induction of apoptosis
in MCF-7 breast adeno-carcinoma cells by PAH-Ag-CPT nanogels was evidenced
by accumulation of late apoptotic cell population. Drug along with
the PAH-Ag NPs were also encapsulated in a pH responsive hydrogel
through in situ gelation at room temperature using acrylic acid as
the cross-linker. The resulting hydrogel released quantitative amounts
of both drug and PAH-Ag NPs over a period of 16 h. The simplicity
of synthesis and ease of drug loading with efficient release render
these NPs a viable candidate for various biomedical applications,
and moreover this synthetic procedure may be extended to other metal
NPs
Conformational Insights into the Lesion and Sequence Effects for Arylamine-Induced Translesion DNA Synthesis: <sup>19</sup>F NMR, Surface Plasmon Resonance, and Primer Kinetic Studies
Adduct-induced DNA damage can affect
transcription efficiency and
DNA replication and repair. We previously investigated the effects
of the 3′-next flanking base (G*C<i><u>T</u></i> vs G*C<i><u>A</u></i>; G*, FABP, <i>N</i>-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl;
FAF, <i>N</i>-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene)
on the conformation of arylamine-DNA lesions in relation to <i>E. coli</i> nucleotide excision repair (Jain, V., Hilton, B., Lin, B., Patnaik, S., Liang, F., Darian, E., Zou, Y., Mackerell, A. D., Jr., and Cho, B. P. (2013) Nucleic Acids Res., 41, 869−880). Here,
we report the differential effects of the same pair of sequences on
DNA replication <i>in vitro</i> by the polymerases exofree
Klenow fragment (Kf-exo<sup>–</sup>) and Dpo4. We obtained
dynamic <sup>19</sup>F NMR spectra for two 19-mer modified templates
during primer elongation: G*C<i>A</i> [d(5′-CTTACCATCG*CAACCATTC-3′)]
and G*C<i>T</i> [d(5′-CTTACCATCG*CTACCATTC-3′)].
We found that lesion stacking is favored in the G*C<i>T</i> sequence compared to the G*C<i>A</i> counterpart. Surface
plasmon resonance binding results showed consistently weaker affinities
for the modified DNA with the binding strength in the order of FABP
> FAF and G*C<i><u>A</u></i> > G*C<i><u>T</u></i>. Primer extension was stalled at
(<i>n</i>) and near (n – 1 and <i>n</i> + 1) the lesion site, and the extent of blockage and the extension
rates across the lesion were influenced by not only the DNA sequences
but also the nature of the adduct’s chemical structure (FAF
vs FABP) and the polymerase employed (Kf-exo<sup>–</sup> vs
Dpo4). Steady-state kinetics analysis with Kf-exo<sup>–</sup> revealed the most dramatic sequence and lesion effects at the lesion
(<i>n</i>) and postinsertion (<i>n</i> + 1) sites,
respectively. Taken together, these results provide insights into
the important role of lesion-induced conformational heterogeneity
in modulating translesion DNA synthesis