The combustion of fossil fuels generates polycyclic aromatic hydrocarbons (PAH) that are ubiquitous and potentially cancer-causing environmental contaminants. PAH are found at toxic waste dumps and superfund sites, in airborne particulates, in our food and water, as well as in tobacco smoke. PAH compounds are believed to contribute to the higher rates of lung and other cancers that have been documented in residents of polluted urban areas and smokers. The PAH are biologically inactive but are metabolically activated to reactive diol epoxides that covalently bind to guanine and adenine in DNA to form stable, pre- mutagenic DNA adducts. Such forms of DNA damage accumulate in tissues of people exposed to PAH- contaminated environments, who are thus at risk of developing respiratory diseases and cancer. Not all DNA adducts are equally threatening to human health. Many of them can be removed by the human DNA repair mechanism called nucleotide excision repair (NER). However, the activity of the NER system is variable: some DNA lesions are slowly repaired, while some others are resistant to NER. The accumulation of such persistent DNA damage enhances the risk of developing diseases of the lung such as asthma, lung cancer, and other disorders. The exact structural features that render certain PAH-DNA adducts NER-resistant, are poorly understood. Among the first mammalian NER factors that recognize and bind to NER substrates is the heterodimeric XPC-RAD23B protein. The identification of the lesions occurs in a two-step (bipartite manner): (1) recognition by XPC-RAD23B, and (2) a subsequent verification step that involves the helicase activity of XPD in TFIIH, a multi-protein NER factor that binds to the XPC-DNA complex. The feasibility of this project is based on a previously developed, extensive library of different PAH-DNA adducts that exhibit the full spectrum of NER activities, from fully resistant to fully susceptible. The objectives are to elucidate the structural features of DNA lesions that abrogate or promote their recognition and repair.
Aim 1 is focused on developing surface plasmon resonance and other methods (footprinting, gel electrophoresis) for studying XPC and TFIIH protein-DNA interactions utilizing a set of well characterized benzo[a]pyrene - diol epoxide guanine lesions (BP-G) in two different base sequence contexts. In one of these, the BP-G lesions are either moderate-to-good NER substrates;in the other sequence, a single nucleotide opposite the BP-G, lesion is missing, and the same BP-G duplexes are fully resistant to NER in human cell extracts.
In Aim 2, these methodologies will be applied to investigate the mechanisms of the initial XPC- RAD23B and TFIIH-DNA binding phenomena utilizing different bulky and small NER-proficient and NER- resistant PAH-guanine and -adenine DNA adducts. Mechanistic insights will be gained by exploring the local thermodynamic destabilization of DNA caused by the lesions using NMR methods, and by molecular modeling and computational techniques.
The human nucleotide excision repair (NER) system represents a critically important first line of defense against bulky polycyclic aromatic hydrocarbons and other carcinogens that are ubiquitous in our urban and industrial environments. A diminished capacity of individuals to repair damaged DNA is believed to be one of the key factors in the etiology of many common cancers. However, it is not known why some bulky DNA lesions can evade the human repair machinery. In this project the structural features that distinguish NER- resistant from NER-proficient DNA lesions will be identified. This information will greatly improve the specificity of existing biomarker-based assessments of human exposure to genotoxic substances in the workplace and polluted environments.
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|Liu, Zhi; Ding, Shuang; Kropachev, Konstantin et al. (2015) Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function. PLoS One 10:e0137124|