Chronic inflammation associated with human exposure to environmental genotoxic chemical contaminants has been implicated in the etiology of human cancers. Chronic inflammation is characterized by an overproduction of reactive oxygen and nitrogen species that cause damage to the cellular DNA that, if not removed by cellular defense mechanisms, may lead to mutations and cancer. The connections between reactive chemical intermediates, their impact on chronic inflammation, and the initiation of cancer, are of great current interest. The primary target of oxidation in DNA is guanine, the most easily damaged nucleobase. DNA repair is a critically important cellular defense mechanisms that plays a key role in safeguarding the genome from the potential deleterious actions of these DNA lesions. The most important basic mechanism of removal of these oxidatively generated forms of DNA damage from the human genome is widely assumed to be the base excision repair (BER) system. However, we have recently discovered that another important cellular defense mechanism, nucleotide excision repair (NER), that normally specializes in the clearing of bulky and DNA helix- distorting adducts, is also capable of removing a number of well known oxidative DNA lesions in human cell extract model systems. In these in vitro systems, the two repair systems compete with one another for the some of the same DNA substrates that have been known to be BER substrates only. However, one mechanism could also hinder the other one, and it is not known whether or how the BER and NER pathways compete with one another since the respective protein levels and availabilities to the DNA lesion substrates are markedly different than in the cell extracts. However, nothing is known about the possible cooperation and competition of these two major repair pathways, BER and NER, in human cells.
In aim 1, the mechanistic aspects of the competition between the BER and NER pathways is quantitatively explored in the controlled environment of cell extracts in which the critical individual BER and NER protein concentrations can be varied at will; the structural requirements for susceptibility of these non-bulky oxidative DNA lesions to NER will be examined by experimental and molecular modeling approaches honed during previous project periods. Preliminary results indicate that the BER and NER pathways do indeed compete with one another in human fibroblasts, and aim 2 is designed to determine the nature of the competition between BER and NER following transfection of site-specifically modified oligonucleotide substrates into human fibroblasts with different genetic backgrounds. A unique library of oxidative DNA lesions has been created that are either repaired by NER only, or by BER only, or by both BER and NER mechanisms. This library of site-specifically modified DNA repair substrates includes 8-oxoguanine, and its deeper oxidation products such as the stereoisomeric spiroimininodihdantoins, guanidinohydantoin, a nitro-imidazole guanine oxidation product, 5?,8-cyclo-2?- deoxypurines, and intrastrand cross-linked guanine-thymine DNA lesions.
Chronic inflammation associated with human exposure to environmental genotoxic chemical contaminants, tobacco smoke, viral infections, ionizing and UV radiation, and other exogenous factors, has been implicated in the etiology of many human cancers. A better understanding of the interplay between base excision repair and nucleotide excision repair of mutagenic oxidatively damaged DNA bases under inflammatory conditions will provide new insights into the competition and cooperation between these two major mammalian DNA repair mechanisms. Ultimately, understanding how these two mechanisms cooperate or hinder one another may need to be considered in therapeutic applications in which the BER and NER pathways contribute to resistance to chemotherapy.
| Merta, Tomasz J; Geacintov, Nicholas E; Shafirovich, Vladimir (2018) Generation of 8-oxo-7,8-dihydroguanine in G-Quadruplexes Models of Human Telomere Sequences by One-electron Oxidation. Photochem Photobiol : |
| Kolbanovskiy, Marina; Chowdhury, Moinuddin A; Nadkarni, Aditi et al. (2017) The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro. Biochemistry 56:3008-3018 |
| Shafirovich, Vladimir; Geacintov, Nicholas E (2017) Removal of oxidatively generated DNA damage by overlapping repair pathways. Free Radic Biol Med 107:53-61 |
