Abstract
Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging 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 excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen 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. This 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.
Highlights
DNA damage is a ubiquitous phenomenon experienced by microbes in the environment
In order to develop a general assay for detecting DNA damage in prokaryotes, the formation of 3Ј-OH DNA ends was monitored in individual cells using TUNEL
Activation of excision DNA repair mechanisms was implied by the requirement of de novo protein synthesis for the formation of 3Ј-OH DNA ends in cells treated with hydrogen peroxide
Summary
DNA damage is a ubiquitous phenomenon experienced by microbes in the environment. Potential DNA-damaging agents include enzymes (e.g., restriction enzymes encoded by addiction modules or bacteriophage) [1, 23, 31], as well as nonenzymatic attacks via physical (e.g., irradiation) [18] and chemical (e.g., secondary metabolites such as antibiotics and reactive oxygen species [ROS]) agents [13]. Indirect DNA repair systems require the recognition and removal of DNA damage (i.e., excision) and the synthesis of new DNA [9]. Indirect DNA repair systems are responsible for fixing most forms of DNA damage [9, 15]. The excision step of indirect DNA repair systems results in single-strand DNA breaks, regardless of the type of original DNA-damaging agent [9, 15]. AP endonucleases nick the strand at the AP site, thereby creating a 3Ј-OH DNA end, which is used as a substrate for synthesis of a new DNA strand to replace the damaged area. Identifying cells with an excess of 3Ј-OH DNA ends would indicate that the cell has experienced DNA damage and is undergoing DNA repair
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