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Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence.

Jain V, Hilton B, Patnaik S, Zou Y, Chiarelli MP, Cho BP - Nucleic Acids Res. (2012)

Bottom Line: Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2).The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization.The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881, USA.

ABSTRACT
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 G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)∼G(1)> G(2)). 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 G(2)∼G(1)> G(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.

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(a) Chromatogram of a reaction mixture between 16-mer NarI sequence (5′-CTCTCG1G2CG3CCATCAC-3′) and an activated FAAF (N-acetoxy-N-2-(acetylamino)-7-fluorofluorene). The mono- (G1, G3, G2), di- and tri-FAAF adducts eluted in the 28–35, 42–60 and 84 min were purified by reversed-phase HPLC (see ‘Materials and Methods’ section for gradient condition); (b) online photodiode array UV/Vis spectra of mono-, di- and tri-FAAF adducts.
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gkr1307-F2: (a) Chromatogram of a reaction mixture between 16-mer NarI sequence (5′-CTCTCG1G2CG3CCATCAC-3′) and an activated FAAF (N-acetoxy-N-2-(acetylamino)-7-fluorofluorene). The mono- (G1, G3, G2), di- and tri-FAAF adducts eluted in the 28–35, 42–60 and 84 min were purified by reversed-phase HPLC (see ‘Materials and Methods’ section for gradient condition); (b) online photodiode array UV/Vis spectra of mono-, di- and tri-FAAF adducts.

Mentions: FAAF-modified 16-mer ODN were prepared using the general procedures described previously (15,27). Briefly, approximately 0.5–1 mg of N-acetoxy-N-2-(acetylamino)-7-fluorofluorene dissolved in absolute ethanol was added drop wise to a sodium citrate buffer (pH 6.0) containing 200–250 ODs of unmodified ODN (5′-CTCTCG1G2CG3CCATCAC-3′) and placed in a shaker for 5 min at 37°C. Figure 2a shows a typical reversed-phase HPLC chromatogram derived from the resulting mixture. The FAAF modified oligomers appearing between 28 and 85 min were separated and purified up to >97% purity by repeated injections. The HPLC system consisted of a Hitachi EZChrom Elite HPLC unit with an L2450 diode array detector and a Phenomenex Luna C18 column (150 × 10 mm, 5.0 µm). We employed a gradient system involving 3–15% acetonitrile for 40 min followed by 15–20% and 20–35% acetonitrile for 20 and 40 min, respectively, in pH 7.0 ammonium acetate buffer (100 mM) with a flow rate of 2.0 ml/min.Figure 2.


Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence.

Jain V, Hilton B, Patnaik S, Zou Y, Chiarelli MP, Cho BP - Nucleic Acids Res. (2012)

(a) Chromatogram of a reaction mixture between 16-mer NarI sequence (5′-CTCTCG1G2CG3CCATCAC-3′) and an activated FAAF (N-acetoxy-N-2-(acetylamino)-7-fluorofluorene). The mono- (G1, G3, G2), di- and tri-FAAF adducts eluted in the 28–35, 42–60 and 84 min were purified by reversed-phase HPLC (see ‘Materials and Methods’ section for gradient condition); (b) online photodiode array UV/Vis spectra of mono-, di- and tri-FAAF adducts.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351159&req=5

gkr1307-F2: (a) Chromatogram of a reaction mixture between 16-mer NarI sequence (5′-CTCTCG1G2CG3CCATCAC-3′) and an activated FAAF (N-acetoxy-N-2-(acetylamino)-7-fluorofluorene). The mono- (G1, G3, G2), di- and tri-FAAF adducts eluted in the 28–35, 42–60 and 84 min were purified by reversed-phase HPLC (see ‘Materials and Methods’ section for gradient condition); (b) online photodiode array UV/Vis spectra of mono-, di- and tri-FAAF adducts.
Mentions: FAAF-modified 16-mer ODN were prepared using the general procedures described previously (15,27). Briefly, approximately 0.5–1 mg of N-acetoxy-N-2-(acetylamino)-7-fluorofluorene dissolved in absolute ethanol was added drop wise to a sodium citrate buffer (pH 6.0) containing 200–250 ODs of unmodified ODN (5′-CTCTCG1G2CG3CCATCAC-3′) and placed in a shaker for 5 min at 37°C. Figure 2a shows a typical reversed-phase HPLC chromatogram derived from the resulting mixture. The FAAF modified oligomers appearing between 28 and 85 min were separated and purified up to >97% purity by repeated injections. The HPLC system consisted of a Hitachi EZChrom Elite HPLC unit with an L2450 diode array detector and a Phenomenex Luna C18 column (150 × 10 mm, 5.0 µm). We employed a gradient system involving 3–15% acetonitrile for 40 min followed by 15–20% and 20–35% acetonitrile for 20 and 40 min, respectively, in pH 7.0 ammonium acetate buffer (100 mM) with a flow rate of 2.0 ml/min.Figure 2.

Bottom Line: Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2).The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization.The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881, USA.

ABSTRACT
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 G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)∼G(1)> G(2)). 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 G(2)∼G(1)> G(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.

Show MeSH
Related in: MedlinePlus