Dna Excision Repair in Lymphocytes Following Photoaffinity Labeling With Ethidium Monoazide

Abstract

Abstract— Irradiation of ethidium monoazide by fluorescent light promotes a chemical decomposition of the azide into a highly reactive nitrene intermediate. Covalent bonding of this electrophile to the DNA in the cell provokes repair of damage which can be monitored by incorporation of [3H]‐thymidine. Human lymphocytes were labeled with [14C]‐ethidium azide and then allowed to undergo DNA repair. Repair incorporation of [3H]‐thymidine showed saturation at 5 µM ethidium azide, but excision of the labeled drug failed to saturate at 20 µM, suggesting that excision and resynthesis are two separate events. Cells were also labeled with the photosensitive drug and/or exposed to UV radiation, and then allowed to undergo a period of DNA repair. The tritium incorporation for the combined insults was less than the sum of the two insults. Quinacrine, progesterone and chloroquine inhibited repair incorporation of [3H]‐thymidine, but had no effect on the excision of the drug from the DNA. After damage by ethidium azide, chromatin was isolated from lymphocytes which had been allowed to repair label with [3H]‐thymidine. Partial digestion of the chromatin with micrococcal nuclease released 80% of the tritium when approximately 40% of the DNA had been hydrolyzed by the enzyme.