We measured the levels of modified DNA bases following ү-irradiation of the halophilic archaeon, Halobacterium sp. str. NRC-1. In this first study to quantify DNA base modification in a prokaryotic system after exposure to ionizing radiation (IR) a direct relationship between number of DNA lesions and IR dose was revealed. Most importantly, our data demonstrated the significant impact of modified DNA bases on cell survival. We also report for the first time in vivo evidence for the scavenging of reactive oxygen species, a product of IR, by intracellular halides in Halobacterium, resulting in increased protection of DNA and proteins from radiation damage. Results also demonstrate that Halobacterium possesses effective DNA repair systems to counter radiation-induced DNA damage.
Ionizing radiation-resistant microorganisms have recently garnered a great deal of attention from scientists seeking to understand the mechanisms underlying the survival abilities of these organisms. Much of the focus has been on mechanisms of cellular repair of radiation-induced DNA damage ranging from repair of DNA strand breaks to repair of modified DNA bases. Whole-genome studies of the response of a variety of microorganisms from all three domains of life, including Deinococcus radiodurans and Halobacterium sp. str. NRC-1 (Halobacterium) to IR, have been used to search for cellular pathways responsible for radiation resistance. These investigations, however, have not revealed any mechanisms that can explain the extreme resistance to IR of these organisms. Indirect damage to DNA through the radiolysis of water producing various reactive species including the highly reactive hydroxyl radical that causes a plethora of DNA damages including base modifications and strand breaks resulting from exposure to IR accounts for the vast majority (approximately 80%) of DNA damages. It has been hypothesized that intracellular salts such as KCl have a role in vivo for radiation-resistant extreme halophiles including Halobacterium that sequester intracellular salts to maintain osmotic balance. This organism is an extreme halophile, requiring 3.5 M to 5 M NaCl (4.3 M NaCl optimal) for growth. Scavenging of radiation-generated hydroxyl radicals by intracellular chloride ions has been proposed to significantly contribute to the radiation resistance of such organisms. This proposal, however, has not been tested in vivo. By measuring DNA damage and its repair in Halobacterium as a model system, the present work, which has been performed in collaboration with Dr. Jocelyne DiRuggiero and her group at Johns Hopkins University, provides evidence that hydroxyl radical scavenging by halides offers significant protection against oxidative DNA damage and that this organism possesses effective DNA repair systems to repair radiation-induced DNA damage. Our findings also have a potential impact on astrobiology with evidence of evaporite deposits containing high concentrations of chloride and bromide in Mars gathered by the Mars Exploration Rovers and the Mars Odyssey orbiter. Lacking an atmosphere and magnetic shield to reduce the surface solar irradiance, microorganisms on the surface of Mars are exposed to far greater levels of UV-C radiation and high-energy radiation than are microorganisms on Earth. Our findings show that the salt environment itself may be a protective factor for potential microbial life on the surface of Mars, indicating that chloride and bromide evaporite deposits showing water modification are excellent areas for surface investigations looking for evidence of life on Mars.
Develop methods and conduct research on the mechanisms of resistance to IR in microorganisms such as Halobacterium.
Research activities and technical approach
We used the model halophile Halobacterium to investigate protection against IR by intracellular halides. Halobacterium, and a majority of extreme halophiles, use KCl as the major compatible solute to counterbalance the high salinity of their natural environment. We measured the level of oxidative damage to DNA bases, the DNA backbone, and protein residues after exposure to IR using in vitro and in vivo methods. Gas chromatog-raphy/mass spectrometry (GC/MS) with isotope-dilution was used to measure modified DNA bases in this organism before and after ү-irradiation at increasing doses. Several typical products of oxidative damage to DNA were identified and quantified. We also used this technique to measure the capability of cells to repair modified DNA bases. Furthermore, protein oxidation was measured by immunodetection of carbonyl groups in Halobacterium protein extracts separated by acrylamide gel electrophoresis.
• Elucidated radiation protection by intracellular halides.
• Developed methods for the measurement of radiation damage to DNA and its repair in radiation-resistant microorganisms.
• Elucidated the significance of the cellular environment in determining radiation resis-tance in microorganisms.