Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as an tibiotics, enzymes, starvation, etc.). The large number of potential DNA-da maging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3'-OH DNA ends, produced by either direct damage or exc ision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleot ide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedur e in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein i sothiocyanate using TdT, Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry, TUNEL was used to measure hydro gen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli, DNA repair systems were implicated in the hydro gen peroxide-dependent generation of 3'-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E, coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. Thi s methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.