Detection of DNA-protein crosslinks (DPCs) by novel direct fluorescence labeling methods: distinct stabilities of aldehyde and radiation-induced DPCs.
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.
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Mahmoud I
MI
Shoulkamy
Toshiaki
T
Nakano
Makiko
M
Ohshima
Ryoichi
R
Hirayama
Akiko
A
Uzawa
Yoshiya
Y
Furusawa
Hiroshi
H
Ide
D000447:Aldehydes / Q000737:chemistry*; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D015687:Cell Hypoxia; D002460:Cell Line; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009374:Neoplasms, Experimental / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D011839:Radiation, Ionizing; D012854:Sister Chromatid Exchange
D000447:Aldehydes; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D011506:Proteins; D004247:DNA; D016650:Fluorescein-5-isothiocyanate