Chronic inflammation caused by diverse chemical, physical and infectious factors increases the risk of malignant cell transformations and the progression of human cancers. The enhanced production of reactive oxygen and nitrogen species (ROS and RNS, respectively) in inflammatory cells leads to oxidative stress that induces an imbalance between oxidative DNA damage and DNA repair. The accumulation of oxidatively generated DNA lesions enhances the mutagenic burden of the cells that can lead to cancer. A primary target of ROS and RNS overproduced at sites of inflammation is guanine, the most easily oxidizable natural nucleic acid base. The primary oxidation step generates guanine radicals that undergo a cascade of chemical reactions with cellular nucleophiles and other substances that lead to the formation of a variety of unstable intermediates and stable genotoxic guanine lesions in DNA. During the previous project period, we have developed new methods and approaches for investigating reaction pathways of guanine radicals, the formation of a variety of stable end-products, and the repair of these lesions by base excision repair (BER) and nucleotide excision repair (NER) mechanisms. The major tools include real time monitoring of the reactions of unstable guanine radical and other intermediates by kinetic laser transient absorption spectroscopy, the isolation and identification of the stable DNA lesions formed by HPLC, LC-MS/MS and MALDI-TOF/MS, and 1D and 2D NMR methods. Major findings are (1) that the one-electron oxidation of guanine is base-sequence dependent, (2) the discovery of a novel guanine-thymine (G*-T*) intrastrand cross-linked lesion that competes with the formation of 8-oxoG, spiroiminodihydantoins (Sp), and other lesions, and (3) that the G*-T* and Sp lesions are substrates for both BER and NER repair mechanisms. The new specific aims build on these preliminary findings with the objectives of clarifying the relationships between the competitive reaction pathways of guanine radicals, the resulting formation of stable guanine lesions, and the susceptibilities of the latter to repair by BER and NER mechanisms.
The specific aims of this project are: 1) determine the effects of base sequence on the distributions of the guanine lesions generated by inflammatory ROS and RNS (peroxynitrite, nitrogen dioxide, superoxide radicals);2) Compare the base sequence-dependent formation of these lesions in naked DNA and nucleosomal DNA;3) Determine the susceptibilities to BER and NER mechanisms of repair using single oxidative guanine lesions in naked and nucleosomal DNA, and monitor oxidative guanine damage and repair in selected repair-proficient and deficient cell lines. A better understanding of DNA damage and repair under conditions simulating oxidative stress should provide a rational basis for discovering new strategies for the prevention and clinical treatments of disease.
Chronic inflammation developed in response to environmental pollutants, tobacco smoke, viral infections, ionizing and UV radiations, and other exogenous factors, is tightly implicated in the etiology of many human cancers. A better understanding of the DNA damage and DNA repair mechanisms under inflammatory conditions will provide a rational basis for discovering new specific biomarkers of oxidative stress, the development of new strategies for the prevention and clinical treatment of inflammatory diseases, and the progression of cancers under inflammatory conditions.
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|Shafirovich, Vladimir; Kropachev, Konstantin; Anderson, Thomas et al. (2016) Base and Nucleotide Excision Repair of Oxidatively Generated Guanine Lesions in DNA. J Biol Chem 291:5309-19|
|Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi et al. (2015) Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways. J Biol Chem 290:14610-7|
|Cadet, Jean; Wagner, J Richard; Shafirovich, Vladimir et al. (2014) One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA. Int J Radiat Biol 90:423-32|
|Rokhlenko, Yekaterina; Cadet, Jean; Geacintov, Nicholas E et al. (2014) Mechanistic aspects of hydration of guanine radical cations in DNA. J Am Chem Soc 136:5956-62|
|Kropachev, Konstantin; Ding, Shuang; Terzidis, Michael A et al. (2014) Structural basis for the recognition of diastereomeric 5',8-cyclo-2'-deoxypurine lesions by the human nucleotide excision repair system. Nucleic Acids Res 42:5020-32|
|Uvaydov, Yuriy; Geacintov, Nicholas E; Shafirovich, Vladimir (2014) Generation of guanine-amino acid cross-links by a free radical combination mechanism. Phys Chem Chem Phys 16:11729-36|
|Khutsishvili, Irine; Zhang, Na; Marky, Luis A et al. (2013) Thermodynamic profiles and nuclear magnetic resonance studies of oligonucleotide duplexes containing single diastereomeric spiroiminodihydantoin lesions. Biochemistry 52:1354-63|
|Madugundu, Guru S; Wagner, J Richard; Cadet, Jean et al. (2013) Generation of Guanine-Thymine Cross-Links in Human Cells by One-Electron Oxidation Mechanisms. Chem Res Toxicol :|
|Rokhlenko, Yekaterina; Geacintov, Nicholas E; Shafirovich, Vladimir (2012) Lifetimes and reaction pathways of guanine radical cations and neutral guanine radicals in an oligonucleotide in aqueous solutions. J Am Chem Soc 134:4955-62|
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