Abstract
DNA-damaging agents from endogenous and environmental sources induce DNA-protein crosslink (DPC) damage, where proteins are irreversibly trapped on DNA through covalent bonds. For the elucidation of the biological effect of DPCs, it is crucial to monitor the induction and intracellular dynamics of DPCs. Several methods have been developed for the detection of DPCs. These include the alkaline elution, nitrocellulose filter binding, sodium dodecyl sulfate-potassium ion precipitation, and single cell gel electrophoresis (comet) methods. Although these methods provided valuable information about the induction and elimination of genomic DPCs induced by various chemical and physical agents, they are all indirect detection methods of DPCs based on the measurement of DNA and not crosslinked proteins, hence offering only semiquantitative information of DPCs. Thus, it would be useful to develop a direct detection method for quantitative analysis of DPCs. In the present study, we have developed a novel direct DPC detection method, where crosslinked proteins were specifically labeled with FITC and assayed for fluorescence. Cells were treated with various aldehydes, typical DPC-inducing agents, and genomic DNA was isolated. The induction and removal of DPCs in cells as well as their in vitro stability were monitored by the FITC-labeling method. These data will be presented in the meeting.
