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Structural and thermodynamic insight into Escherichia coli UvrABC-mediated incision of cluster diacetylaminofluorene adducts on the NarI sequence.

ABSTRACT: Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1*G2CG3*CC-3' (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.

SUBMITTER: Jain V

PROVIDER: S-EPMC3917139 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Structural and thermodynamic insight into Escherichia coli UvrABC-mediated incision of cluster diacetylaminofluorene adducts on the NarI sequence.

Jain Vipin V Hilton Benjamin B Lin Bin B Jain Anshu A MacKerell Alexander D AD Zou Yue Y Cho Bongsup P BP

Chemical research in toxicology 20130725 8

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1 ...[more]

PMID: 23841451

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Project description:Nucleotide excision repair (NER) plays an important role in maintaining the integrity of DNA by removing various types of bulky or distorting DNA adducts in both prokaryotic and eukaryotic cells. In Escherichia coli, the excision repair proteins UvrA, UvrB, and UvrC recognize and incise the bulky DNA damages induced by UV light and chemical carcinogens. In this process, when a putative lesion in DNA is identified initially by UvrA, a subsequent strand opening is carried out by UvrB that not only ensures that the distortion is indeed due to a damaged nucleotide but also recognizes the chemical structure of the modified nucleotides with varying efficiencies. UvrB also recruits UvrC that catalyzes both the 3'- and the 5'-incisions. Herein, we examined the interaction of UvrABC with a DNA substrate containing a single G[8,5-Me]T cross-link and compared it with T[6,4]T (the 6-4 pyrimidine-pyrimidone photoproduct) and the C8 guanine adduct of N-acetyl-2-aminofluorene (AAF). The intrastrand vicinal cross-link G[8,5-Me]T containing a covalent bond between the C8 position of guanine and the 5-methyl carbon of the 3'-thymine is formed by X-radiation, while T[6,4]T is a vicinal cross-link induced by the UV light. We also selected the AAF adduct for comparison because it represents a highly distorting monoadduct containing a covalent linkage at the C8 position of guanine. The dissociation constants (K(d)) for UvrA protein binding to DNA substrates containing the G[8,5-Me]T, T[6,4]T, and AAF adducts, as determined by gel mobility shift assays, were 3.1 +/- 1.3, 2.8 +/- 0.9, and 8.2 +/- 1.9, respectively. Although UvrA had a considerably higher affinity for G[8,5-Me]T than for the AAF adduct, the G[8,5-Me]T intrastrand cross-link was incised by UvrABC much less efficiently than the T[6,4]T intrastrand cross-link and the AAF adduct. Similar incision results also were obtained with the DNA substrates containing the adducts in a six-nucleotide bubble, indicating that the inefficient incision of G[8,5-Me]T cross-link by UvrABC was probably due to the lack of efficient recognition of the adduct by UvrB at the second step of DNA damage recognition in the E. coli NER. Indeed, as compared to T[6,4]T and AAF substrates, which clearly showed UvrB-DNA complex formation, very little UvrB complex was detectable with the G[8,5-Me]T substrate. Our result suggests that G[8,5-Me]T intrastrand cross-link is more resistant to excision repair in comparison with the T[6,4]T and AAF adducts and thus will likely persist longer in E. coli cells.

Dataset Information

Structural and thermodynamic insight into Escherichia coli UvrABC-mediated incision of cluster diacetylaminofluorene adducts on the NarI sequence.

Publications

Structural and thermodynamic insight into Escherichia coli UvrABC-mediated incision of cluster diacetylaminofluorene adducts on the NarI sequence.

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