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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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Absolute percent incision rates of (a) FAAF and (b) FAF–NarI duplexes modified at G1, G2 and G3; (c) percent incision rates histogram of FAF and FAAF at different positions relative to NarI–G3 FAAF as 100%.
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gkr1307-F6: Absolute percent incision rates of (a) FAAF and (b) FAF–NarI duplexes modified at G1, G2 and G3; (c) percent incision rates histogram of FAF and FAAF at different positions relative to NarI–G3 FAAF as 100%.

Mentions: Figure 6 shows the kinetic assay results, in which 55-mer FAAF-modified DNA duplex substrates were incised by UvrABC nuclease. These substrates were radioactively labeled at the 5′-end of the adducted strand. The major incision products can be seen as 18-mer (NarI–G1), 19-mer (NarI–G2) or 21-mer (NarI–G3) separated on a urea–PAGE gel under denaturing conditions (Supplementary Figure S12). The incision occurred at the eighth phosphate bond 5′ to the modified nucleotide, which is consistent with the previously reported results of UvrABC incision (11,32).Figure 6.


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)

Absolute percent incision rates of (a) FAAF and (b) FAF–NarI duplexes modified at G1, G2 and G3; (c) percent incision rates histogram of FAF and FAAF at different positions relative to NarI–G3 FAAF as 100%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1307-F6: Absolute percent incision rates of (a) FAAF and (b) FAF–NarI duplexes modified at G1, G2 and G3; (c) percent incision rates histogram of FAF and FAAF at different positions relative to NarI–G3 FAAF as 100%.
Mentions: Figure 6 shows the kinetic assay results, in which 55-mer FAAF-modified DNA duplex substrates were incised by UvrABC nuclease. These substrates were radioactively labeled at the 5′-end of the adducted strand. The major incision products can be seen as 18-mer (NarI–G1), 19-mer (NarI–G2) or 21-mer (NarI–G3) separated on a urea–PAGE gel under denaturing conditions (Supplementary Figure S12). The incision occurred at the eighth phosphate bond 5′ to the modified nucleotide, which is consistent with the previously reported results of UvrABC incision (11,32).Figure 6.

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