Mehanizmi izjemne odpornosti proti radioaktivnemu sevanju pri prokariontih

Introduction

Extremophiles are organism that live, metabolize and reproduce to human extreme conditions. We define a variety of extreme conditions such as high or low temperatures, pH, salinity, pressure and radiation. Scientists find this organisms extraordinary, especially their metabolism, mechanisms of DNA repair and protein antioxidants. The most impressive extremophile organisms are those resistant to high levels of ionizing radiation. Bacterium Deinococcus radiodurans can survive exposures as high as 15 kGy (Gray is an SI unit for absorbed radiation dose), while for humans a lethal dose is around 10 Gy and average bacterium around 200 Gy. Ionizing radiation causes severe damage on living cells such as formation of ROS, DNA single and double strand brakes, DNA extensive base modifications. Evidently some bacterium have mechanism that fight radiation damage. Due to lack of radioprotectors this bacterium ability rises high interest as a potential suppressor of radiation effects. Suppressing side effect on healthy cells in cancer radiations treatments would have enormous benefit as higher radiation doses would be allowed with better anticancer effect.

Analysis

This highly complex mechanism can’t be a single standing one. Because of its complicity a series of mechanism need to interact to repair and resist radiation. The mechanism would have to consist of a more efficient DNA repair mechanism (In comparison with radiation intolerant species), nucleotide condensation, DNA and protein antioxidant system, protein recovery and lipid dynamics. It’s important to mention that radiotolerant strains are not phylogenetically related and that a universal mechanism doesn’t exist. Because of their diversity it is believed that radiotoleration was acquired by different strains during evolution. We can’t study just D. radiodurans and conclude that the mechanism is broad for resistant bacterium. There is a bunch of different genes and proteins that have roles in their domains. We focus mostly on D. radiodurans because it is the most resistant and studied bacterium (Research led by Croatian scientist Miroslav Radman, fully sequenced genome) and with sequence analysis find correspondence with other resistant bacterium. D. radiodurans can be found in organic-rich environments and harsh environments like deserts and rocks. It is believed that D. radiodurans resistance to radiation stress was as a byproduct to dehydration adaptation. Other studies state that D. radiodurans could be Martian descendant, as the earth lacks radiation levels that could explain D. radiodurans high resistance. It is interesting to mention that radioresistance was developed under laboratory conditions on sensitive E.coli. Cycles of radiation lead to genetic mutations in key mechanisms such as recA protein and higher ratio of Mn/Fe intercellular ions. This alteration are similar to that of D. radiodurans which we will explain in the following paragraph.

Protein antioxidant system

Protein oxidation causes covalent modifications on proteins leading to changes in protein physical and chemical properties (conformation, structure, solubility, proteolysis, activity,) which can lead to cell death and mutations. Carbonylation (introduction of carbon monoxide) is the most common oxidative modification, used as a biomarker for protein oxidation. Protein oxidation stress caused by oxygen species: hydroxyl radicals (OH•), superoxide radicals (O2-•), and hydrogen peroxide (H2O2). Deinococcus radiodurans is resistant to all three oxygen species by an enzymatic and nonenzymatic antioxidant system. Enzymatic system is based on higher levels of catalases (H2O2), superoxide dismutases (O2-•) and peroxidases (H2O2) than in radiosensitive species. Apart from the primary enzymes, D. radiodurans has more oxidative defense proteins in the form of: glutaredoxin, thioredoxin, thioredoxin reductase and alkyl hydroperoxide reductase. Dps proteins (Ferritin-like protein, iron releasing/storage) protect from oxidative damage by binding DNA and reducing H2O2. Nonenzymatic defense is based on carotenoids and manganese complexes. Carotenoids play an important role in oxidative stress protection, but their presence isn’t necessarily needed as they don’t contribute on radiation stress. D. radiodurans has a high intracellular manganese concentration. Manganese forms complexes with cellular metabolites, which is a very effective defense. It has the ability to replace Fe in proteins protecting from fenton (H2O2 iron catalyzed oxidation reaction) driven protein oxidation. It was found that the high ratio of intercellular Mn/Fe is linked to low protein oxidation and high radiation resistance. Mn mostly forms complexes with orthophosphate (O2-•) and peptidase. Mn defense system is effective in protecting DNA repair proteins, protein recovery and oxidation, but ineffective against DSBs.

DNA repair mechanism

Precise and accurate DNA repair mechanism is of great importance for bacterial genome repairs caused by radiation stress. Sequence analysis in DNA repair hasn’t found a single protein conserved in all species. Protein recA is detected the highest percentage in the sequence analysis. RecA has a pivotal role in D. radiodurans DNA repair. RecA-mediated homologous recombination is crucial for DSB. RecA also interacts with Holliday junction resolvasome RuvABC. Apart from recA pathways, bacterium have a recA independent mechanism, non-homologous end joining (NHEJ). One third of the mechanism doesn’t require recA. The mechanism is based on proteins like DdrA, DdrB, SSB and RadA. RecA dependent/independent mechanisms are believed to interact with one another

Nucleotide condensation

D. radiodurans has a more condensed genomic DNA to maintain DNA linearity and protection of free radicals formed in the cytoplasm. The ability of DNA linearity in the presence of DNA breaks highly increase repair possibility by not allowing DNA fragments to diffuse to higher distances.

Histone like proteins

Histone like proteins (HU) are bacterial proteins that share a similar role as eukaryotic histones, the ability of DNA binding. HU can control gene expression and nucleotide organization. The bacteria constantly have high concentrations of HU present, so higher levels are not needed during radiation stress. HU doesn’t have the ability to bind on DNA fragments so it had to adapt a particular structure during evolution that would enable DNA fragment binding. HU binds to a four way DNA junction, and stabilizes recombination intermediates.

Lipid dynamics

Bacterial membranes have modified lipid, protein and carbohydrate compositions to survive in rough environments that were mentioned above. The membrane is the first line of defense from radiation. Although there isn’t a lipid structure that can’t block radiation, its ability to sustain radiation damage, adapt and integrate with other mechanisms separates them from radio intolerant organisms. Radiation can cause oxidation of membranes proteins and puncture. Although the mechanism isn’t still known it is believed that the protein antioxidant system protects membrane proteins which is of great importance because of the signal pathways, transportation and defense roles that the proteins have. Some changes in the fatty acid composition were found leading to change in membrane fluidity which is believed to have a signal role. Another possible resistance advantage is the presence of the S-layer D. radiodurans membrane. The loss of the S-layer lead to higher stress damage.

References

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2. Slade D., et all., Oxidative Stress Resistance in Deinococcus radiodurans, Microbiology and molecular biology reviews, 2011., Vol. 75.

3. Munteanu A., et all., Recent progress in understanding the molecular mechanisms of radioresistance in Deinococcus bacteria, Extremophiles, 2015., Vol. 19.

4. Tian B., et all., Proteomic analysis of membrane proteins from a radioresistant and moderate thermophilic bacterium Deinococcus geothermalis, Molecular BioSystems, 2010., Vol. 10.