Abstract
ABSTRACTOne of the most often used methods to monitor DNA repair in vivo is the host cell reactivation assay (HCR). It is based on the recovery of in vitro damaged plasmids after transfection in host cells. However, it is not clear to what extent plasmid molecules were degraded in the cells and whether they were packed with histones to form chromatin. Since these questions are important to evaluate the results obtained with HCR, in the present paper we studied the fate of the plasmid pEGFP-N1 after transfection in HEK 293 cells. To this end nuclei isolated from cells transfected with native and trioxsalen crosslinked pEGFP-N1 were digested with micrococcal nuclease (MNase) and DNA was subjected to electrophoresis. Southern blots were prepared and probed with digoxigenin-labeled plasmid DNA to reveal the plasmid DNA digestion pattern. Our results showed that nucleosome-like particles were formed on both native and damaged plasmid DNA after transfection. However, the nucleosome ladders were anomalous compared to the ladders generated by digestion of bulk cellular chromatin.
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