Quantitation of Apurinic/Apyrimidinic Sites in Isolated DNA and in Mammalian Tissue with a Reduced Level of Artifacts

Abstract

The apurinic/apyrimidinic (AP) site is a common lesion of DNA damage. The levels of AP sites reported in the literature range widely, which is primarily due to the artefactual generation or loss of AP sites during processing of the DNA. Herein, we have developed a method to quantitate AP sites with a largely reduced level of artifacts by derivatizing AP sites before DNA isolation. A rapid digestion of nuclear protein was performed to minimize enzymatic DNA repair, followed by direct derivatization of AP sites in the nuclear lysate with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA), yielding an oxime derivative that is stable through subsequent DNA processing steps. Quantitation was done using highly selective and sensitive liquid chromatography-tandem mass spectrometry, with a limit of quantitation at 2.2 lesions per 10 8 nucleotides (nts, 0.9 fmol on column). The method was applied in vivo to measure AP sites in rats undergoing oxidative stress [liver: 3.31 ± 0.47/10 7 nts (dosed) vs 0.91 ± 0.06/10 7 nts (control), kidney: 1.60 ± 0.07/10 7 nts (dosed) vs 1.13 ± 0.12/10 7 nts (control)]. The basal AP level was significantly lower than literature values. The method was also used to measure AP sites induced by the chemotherapeutic nitrogen mustard in vitro.

DNA Crosslinkomics: A Tool for the Comprehensive Assessment of Interstrand Crosslinks Using High Resolution Mass Spectrometry

“…Artifactual formation of AP sites during DNA isolation could derive from the BER enzyme activity, spontaneous depurination at high temperature and/or low pH, and β -elimination. 37 These artifactually derived AP sites might result in the formation of further AP site-associated DNA crosslinks.…”

How (5′S) and (5′R) 5′,8-Cyclo-2′-Deoxypurines Affect Base Excision Repair of Clustered DNA Damage in Nuclear Extracts of xrs5 Cells? A Biochemical Study

“…The most common ones have the chemical structure of nucleobases modified (alkylation, deamination, oxidation), which may lead to helix distortion [ 4 ]. One of the most frequently occurring lesions is 8-oxo-7,8-dihydroguanine (8-oxo-dG; 1 in every 10 6 deoxyguanosine) or apurinic/apyrimidinic sites (AP sites), which are chemically unstable and thus highly mutagenic (1.6–3.3 in every 10 7 nucleotides in mammalian tissues) [ 6 , 7 ]. Isolated DNA lesions are detected by glycosylases, such as evolutionary conserved uracil-DNA glycosylase (UDG).…”

Ultrasensitive recognition of AP sites in DNA at the single-cell level: one molecular rotor sequentially self-regulated to form multiple different stable conformations

“…9,10 Thus, developing active substrate molecules that can take part in the relevant chemical reactions induced by the AP site is immensely conducive for such site-specic recognition in a DNA sequence. 11,12 To date, combined with nuclear magnetic resonance (NMR) spectrometry, 13,14 mass spectrometry 15,16 and optical techniques, [17][18][19][20] some substrate molecules have been successfully implemented for quantifying AP sites in DNA. [13][14][15][16][17][18][19][20] NMR spectrometry for detecting AP sites is characterized by highly selective signals.…”

“…13,14 Other substrate molecules, under the condition of mass spectrometry, can emit a highly sensitive mass spectrometry signal but cannot in situ detect the AP sites in living cells. 15,16 Still other substrate molecules with optical signals, such as UV-visible and uorescence signals, only exhibit a single optical signal change at one wavelength. [17][18][19][20] Thus, the stability of the recognition signal (especially uorescence recognition signal) for the AP sites is relatively poor in living cells.…”