Detection of DNA–protein crosslinks (DPCs) by novel direct fluorescence labeling methods: distinct stabilities of aldehyde and radiation-induced DPCs

Abstract

Proteins are covalently trapped on DNA to form DNA–protein crosslinks (DPCs) when cells are exposed to DNA-damaging agents. DPCs interfere with many aspects of DNA transactions. The current DPC detection methods indirectly measure crosslinked proteins (CLPs) through DNA tethered to proteins. However, a major drawback of such methods is the non-linear relationship between the amounts of DNA and CLPs, which makes quantitative data interpretation difficult. Here we developed novel methods of DPC detection based on direct CLP measurement, whereby CLPs in DNA isolated from cells are labeled with fluorescein isothiocyanate (FITC) and quantified by fluorometry or western blotting using anti-FITC antibodies. Both formats successfully monitored the induction and elimination of DPCs in cultured cells exposed to aldehydes and mouse tumors exposed to ionizing radiation (carbon-ion beams). The fluorometric and western blotting formats require 30 and 0.3 μg of DNA, respectively. Analyses of the isolated genomic DPCs revealed that both aldehydes and ionizing radiation produce two types of DPC with distinct stabilities. The stable components of aldehyde-induced DPCs have half-lives of up to days. Interestingly, that of radiation-induced DPCs has an infinite half-life, suggesting that the stable DPC component exerts a profound effect on DNA transactions over many cell cycles.

Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage

“…Moreover, they cannot differentiate between exogenous and endogenous formaldehyde-induced DPCs. Other studies have shown a rapid decline of the formaldehyde-induced DPCs in cell cultures, and no accumulation of DPCs was observed in rats after repeated exposure (Casanova et al, 1994;Grafstrom et al, 1984;Shoulkamy et al, 2012). Further evidence indicates that the major portions of formaldehyde-induced DPCs are lost from lymphocytes through spontaneous (Dunnett, 1964).…”

Measurement of Endogenous versus Exogenous Formaldehyde–Induced DNA–Protein Crosslinks in Animal Tissues by Stable Isotope Labeling and Ultrasensitive Mass Spectrometry

“…Accurate measurement of DPCs is complicated by two critical issues. First, covalent DPCs must be completely isolated from non-covalent DNA-protein complexes, since the latter are present in a clear excess over the former throughout the genome (16). Secondly, DPCs need to be measured with structural specificity, given that there are numerous lesions that complicate the background of DPCs resulting from both endogenous and exogenous electrophiles.…”

“…Secondly, DPCs need to be measured with structural specificity, given that there are numerous lesions that complicate the background of DPCs resulting from both endogenous and exogenous electrophiles. However, such stringent measurements of DPCs have not been possible using previously available DPC detection techniques, such as SDS/KCl precipitation, phenol-chloroform extraction, nitrocellulose filter binding, comet assay, alkaline elution, and CsCl density gradient centrifugation, all of which are nonselective (16,17). Recently, mass spectrometry-based methods have emerged as powerful tools for chemical analysis of digested DPCs (11).…”

DNA–protein crosslink repair

“…Because DPCs apparently pose a less-imminent threat to cellular survival than chromosome breaks, hypoxic cells are more resistant to ionizing radiation. The inverse relationship between DPCs and DSB formation is also observed following irradiation of hypoxic and normoxic tumours 20 , and is thought to be the cause of the less-effective killing of cells within the hypoxic areas of a tumour during cancer r adiotherapy 20 (FIG. 1c).…”