Determination of Apurinic/Apyrimidinic Lesions in DNA with High-Performance Liquid Chromatography and Tandem Mass Spectrometry

Abstract

A new method has been developed to accurately measure apurinic and apyrimidinic (AP) DNA damage sites, which are lesions in DNA formed by loss of a nucleobase from oxidative stress or carcinogen adducts. If AP sites are left unrepaired (or if improperly repaired), these sites can lead to DNA mutations that may ultimately result in the formation of cancer. Hence, detection of AP sites may provide a useful indicator of exposure and susceptibility to chemical carcinogens and oxidative stress. AP detection is currently accomplished by immunodetection methods using an aldehyde reactive probe [Nakamura, J., Walker, V. E., Upton, P. B., Chiang, S.-Y., Kow, Y. W., and Swenberg, J. A. (1998) Cancer Res. 58, 222-225; Atamna, H., Cheung, I., and Ames, B. N. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 686-691]; however, these approaches lack the specificity required for unequivocal identification of the AP site. Therefore, we have developed an accurate method based on mass spectrometry detection of AP sites from AP DNA that have been prelabeled with O-4-nitrobenzylhydroxylamine (NBHA). Once labeled and once the excess labeling agent has been removed, enzymatic digestion of DNA to monomeric subunits can be accomplished, followed by isolation and detection with high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Optimization and validation of the experimental procedures and detection limits have been established using a model DNA oligomer (11-mer) containing uracil. Enzymatic removal of uracil with uracil glycosylase generates well-defined AP sites in both single- and double-stranded DNA. The addition of NBHA labels the AP site in the oligomer, creating a labeled 11-mer. HPLC-ESI-MS/MS in the negative ionization mode was used to monitor and confirm binding of NBHA to the AP oligomer. The NBHA-tagged oligomer underwent endo- and exonuclease digestion to the 5'-deoxyribose monophosphate (5'-dRp) level, thereby releasing free 5'-dRp-NBHA. The 5'-dRp-NBHA product was partially purified by solid phase extraction and quantified by LC-MS/MS using several transitions of the deprotonated molecule ([M - H]- at m/z 363) and isotopically labeled 5'-dRp-NBHA as an internal standard. Further experiments with 5',3'-bisphosphate-deoxyribose and heat/acid-treated calf thymus DNA showed similar labeling, digestion, and detection results. Initial results show a quantification limit with 100 mug of DNA to be 100 fmol (three abasic sites per 10(7) bases).

The impact of acute temperature stress on hemocytes of invasive and native mussels (Mytilus galloprovincialisandM. californianus): DNA damage, membrane integrity, apoptosis and signalling pathways

“…the production of reactive oxygen species, and from physical and chemical environmental stressors. DNA damage due to combinations of environmental factors and normal metabolic processes occurs at a high rate in human cells and increases under environmental stress (Roberts et al, 2006;Prasad et al, 2011). Single base lesions causing SSBs are the most common forms of DNA damage (Roberts et al, 2006;Prasad et al, 2011).…”

“…DNA damage due to combinations of environmental factors and normal metabolic processes occurs at a high rate in human cells and increases under environmental stress (Roberts et al, 2006;Prasad et al, 2011). Single base lesions causing SSBs are the most common forms of DNA damage (Roberts et al, 2006;Prasad et al, 2011). Although SSBs can generally be repaired, they are known to be the initial signal for activating the SOS repair response in bacteria (Craig and Roberts, 1981) and to act as the initial signal for DNA damage responses in eukaryotic cells (Li and Deshaies, 1993).…”

Unambiguous structural elucidation of base‐modified purine nucleosides using NMR

“…In our recent work, [12] we have developed a novel method to prepare oligodeoxyribonucleotides (oligomers) containing modified purines. One of the key compounds was N1-dinitrophenyldeoxyinosine 4 that was prepared from 3 and subsequently converted into various purine-modified nucleosides (5)(6)(7)(8) as shown in Scheme 2. Here, we use the key compound 4 as a typical example to highlight some of the NMR characteristics for general purine-modified nucleosides.…”

“…Therefore, for a majority of DNA-related chemical studies, authentic samples are usually in the form of nucleosides, e.g., as standards in nucleoside composition analysis to identify base-modified moieties by HPLC. [6] Depurination methods have been used [7] in which modified DNA is treated with acid to release the purines to be analysed. However, in some cases this method cannot be used since the acidic conditions used for depurination are frequently too harsh for modified bases to survive.…”

A review of recent experiments on step-to-step “hand-off” of the DNA intermediates in mammalian base excision repair pathways

“…DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1000 to 1000000 molecular lesions per cell per day [4–6]. While this constitutes only a small portion of the human genome's approximately 6 billion bases (3 billion base pairs), misrepaired or unrepaired lesions in critical genes (such as tumor suppression genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation and other adverse conditions.…”