ABSTRACT
Rubrobacter radiotolerans is an extremely radioresistant bacterium. It exhibits higher resistance than the well-known radioresistant bacterium Deinococcus radiodurans, but the molecular mechanisms responsible for the radio-resistance of R. radiotolerans remain unknown. In the present study, we have demonstrated the presence of a novel DNA repair enzyme in R. radiotolerans cells that recognizes radiation-induced DNA damages such as thymine glycol, urea residues, and abasic sites. The enzyme was purified from the crude cell extract by a series of chromatography to an apparent physical homogeneity. The purified enzyme showed a single band with a molecular mass of approximately 40 kDa in SDS-polyacrylamide gel electrophoresis, and was designated as R-endonuclease. R-Endonuclease exhibited repair activity for thymine glycol, urea residues, and abasic sites present in plasmid DNA, but did not act on intact DNA, UV-irradiated DNA and DNA containing reduced abasic sites. The substrate specificity together with the salt and pH optima suggests that R-endonuclease is a functional homolog of endonuclease III of Escherichia coli.
Subject(s)
Arthrobacter/enzymology , Bacterial Proteins/isolation & purification , DNA Ligases/isolation & purification , DNA Repair/genetics , DNA, Bacterial/genetics , Deoxyribonuclease (Pyrimidine Dimer) , Endonucleases/isolation & purification , Escherichia coli Proteins , Radiation Tolerance/genetics , Apurinic Acid/analysis , Arthrobacter/genetics , Arthrobacter/radiation effects , Bacterial Proteins/genetics , DNA Damage , DNA Ligases/genetics , DNA, Bacterial/radiation effects , Electrophoresis, Polyacrylamide Gel , Endodeoxyribonucleases/metabolism , Endonucleases/chemistry , Endonucleases/genetics , Escherichia coli , Radon , Substrate Specificity , Thymine/analogs & derivatives , Thymine/metabolism , Urea/metabolism , Water MicrobiologyABSTRACT
Halobacterium salinarium, a member of the extremely halophilic archaebacteria, contains a C50-carotenoid namely bacterioruberin. We have previously reported the high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV). In this study, we have examined whether bacterioruberin and the highly concentrated salts in this bacterium play protective roles against the lethal actions of ionizing radiation, UV, hydrogen peroxide, and mitomycin-C (MMC). The colourless mutant of H. salinarium deficient in bacterioruberin was more sensitive than the red-pigmented wild-type to all tested DNA-damaging agents except MMC. Circular dichroism (CD) spectra of H. salinarium chromosomal DNA at various concentrations of KCl (0-3.5 M) were similar to that of B-DNA, indicating that no conformational changes occurred as a result of high salt concentrations. However, DNA strand-breaks induced by ionizing radiation were significantly reduced by the presence of either bacterioruberin or concentrated KCl, presumably due to scavenging of free radicals. These results suggest that bacterioruberin and intracellular KCl of H. salinarium protect this organism against the lethal effects of oxidative DNA-damaging agents.
Subject(s)
Carotenoids/physiology , DNA/radiation effects , Halobacterium salinarum/physiology , Potassium Chloride/pharmacology , DNA/drug effects , Halobacterium salinarum/drug effects , Halobacterium salinarum/radiation effectsABSTRACT
Lethal effects of 60Co gamma-rays, UV light, and mitomycin C on two kinds of bacteria, Halobacterium salinarium which grows in highly concentrated salt media and Thiobacillus intermedius which requires reduced sulfur compounds, were studied and compared with those on Escherichia coli B/r. D37 values for H. salinarium, T. intermedius and E. coli B/r were 393, 150, and 92 Gy, respectively, by exposure to 60Co gamma-rays. They were 212, 38, and 10 J/m2, respectively, by exposure to UV light and 2.36, 0.25, and 0.53 microgram/ml/h, respectively, by exposure to mitomycin C. Against these agents, H. salinarium was much more resistant than T. intermedius and E. coli B/r.
Subject(s)
Halobacterium/radiation effects , Thiobacillus/radiation effects , Cobalt Radioisotopes , Escherichia coli/drug effects , Escherichia coli/radiation effects , Gamma Rays , Halobacterium/drug effects , Mitomycin/pharmacology , Radiation Tolerance , Thiobacillus/drug effects , Ultraviolet RaysABSTRACT
High concentration salt effects on the structure and radiation-induced damages of DNA were studied to elucidate the biochemical mechanism of the resistance of halophilic H. salinarium against DNA damaging agents. High concentration of KCl did not induce significant conformational changes in H. salinarium chromosomal DNA, but exhibited an extensive protective effect on the radiation-induced single-strand breaks of plasmid DNA.
