Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

Abstract

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.

Dynamics of a Benzo[a]pyrene-derived Guanine DNA Lesion in TGT and CGC Sequence Contexts: Enhanced Mobility in TGT Explains Conformational Heterogeneity, Flexible Bending, and Greater Susceptibility to Nucleotide Excision Repair

“…58 This structure emphasizes the importance of duplex destabilization by the lesion in facilitating recognition. 59 …”

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

“…Eight XP complementation groups are known, of which seven are caused by mutations in genes involved in the NER process (3,4). NER recognizes a large array of diverse lesions (5)(6)(7)(8)(9). It repairs UVand cisplatin-induced intrastrand crosslinks (10,11).…”

“…The NER system also repairs a wide variety of single base bulky DNA adducts formed by environmental carcinogens. NER is thus the most versatile known DNA repair system (5,8,9).…”

“…Although the function of most XP-proteins within the NER process is understood, the precise role of XPA has remained unclear XPA is known to interact with other NER proteins such as RPA, TFIIH, and ERCC1 (30)(31)(32)(33) suggesting XPA to be a NER scaffold protein (3). Furthermore, detailed biochemical experiments showed that XPA binds to kinked DNA structures (34) and to DNA duplexes containing certain types of DNA lesions, such as cisplatin (10,35,36) adducts and bulky adducts (5,37,38). XPA was found to form a homodimer (38), and it was demonstrated that it forms a 2:1 complex with the respective DNA (37).…”

Integrating Field Analyses with Laboratory Exposures to Assess Ecosystems Health

“…While PAH-DNA adducts represent a type of DNA damage, they can be converted into heritable mutations by misrepair or faulty DNA synthesis (Watanabe et al, 1985;Rodriguez & Loechler, 1995). Bay-or fjord-region diol epoxide-DNA adducts are repaired by nucleotide excision repair (Geacintov et al, 2002). Numerous examples have shown that bay-and fjord-region diol epoxides of PAHs are mutagenic in bacteria, cause damage to DNA or induce chromosomal damage in human and mammalian cells in culture and induce skin, lung or liver tumours in mice, similarly to the parent PAH.…”