Identification and evaluation of the role of the manganese efflux protein in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background  

Deinococcus radiodurans accumulates high levels of manganese ions, and this is believed to be correlated with the radiation resistance ability of this microorganism. However, the maintenance of manganese ion homeostasis in D. radiodurans remains to be investigated.     

A ring-like nucleoid is not necessary for radioresistance in the <Emphasis Type="Italic">Deinococcaceae</Emphasis>

Background  

Transmission electron microscopy images of Deinococcus radiodurans R1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. Since extreme radioresistance is characteristic of six other species of Deinococcus, we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic DNA of each of these species exhibit similar structures.     

RecA Proteins from <Emphasis Type="Italic">Deinococcus geothermalis</Emphasis> and <Emphasis Type="Italic">Deinococcus murrayi</Emphasis> - Cloning,Purification and Biochemical Characterisation

Background  

Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown.     

The single-stranded DNA-binding protein of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background

Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.

Results

A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSB_Dr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.

Conclusions

The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

     

Comparative genomics of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>: divergent routes of adaptation to thermophily and radiation resistance

Background  

Thermus thermophilus and Deinococcus radiodurans belong to a distinct bacterial clade but have remarkably different phenotypes. T. thermophilus is a thermophile, which is relatively sensitive to ionizing radiation and desiccation, whereas D. radiodurans is a mesophile, which is highly radiation- and desiccation-resistant. Here we present an in-depth comparison of the genomes of these two related but differently adapted bacteria.     

Roles of PprA,IrrE, and RecA in the resistance of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> to germicidal and environmentally relevant UV radiation

To study the role of different DNA repair genes in the resistance of Deinococcus radiodurans to mono- and polychromatic UV radiation, wild-type strain and knockout mutants in RecA, PprA, and IrrE of D. radiodurans were irradiated with UV-C (254 nm), UV-(A + B) (280–400 nm) and UV-A (315–400 nm) radiation, and survival was monitored. The strain deficient in recA was highly sensitive to UV-C radiation compared to the wild-type, but showed no loss of resistance against irradiation with UV-(A + B) and UV-A, while pprA and irrE-deficient strains exhibited elevated sensitivity to UV-A and UV-(A + B) radiation. These results suggest that the repair of DNA double-strand breaks is essential after treatment with highly energetic UV-C radiation, whereas protection from oxidative stress may play a greater role in resistance to environmentally relevant UV radiation.     

RecA protein assures fidelity of DNA repair and genome stability in Deinococcus radiodurans

Deinococcus radiodurans is one of the most radiation-resistant organisms known. It can repair hundreds of radiation-induced double-strand DNA breaks without loss of viability. Genome reassembly in heavily irradiated D. radiodurans is considered to be an error-free process since no genome rearrangements were detected after post-irradiation repair. Here, we describe for the first time conditions that frequently cause erroneous chromosomal assemblies. Gross chromosomal rearrangements have been detected in recA mutant cells that survived exposure to 5 kGy γ-radiation. The recA mutants are prone also to spontaneous DNA rearrangements during normal exponential growth. Some insertion sequences have been identified as dispersed genomic homology blocks that can mediate DNA rearrangements. Whereas the wild-type D. radiodurans appears to repair accurately its genome shattered by 5 kGy γ-radiation, extremely high γ-doses, e.g., 25 kGy, produce frequent genome rearrangements among survivors. Our results show that the RecA protein is quintessential for the fidelity of repair of both spontaneous and γ-radiation-induced DNA breaks and, consequently, for genome stability in D. radiodurans. The mechanisms of decreased genome stability in the absence of RecA are discussed.     

DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks

Background  

DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

Effects of radiation quality on interactions between oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation damages DNA and also induces oxidative stress, which can affect the function of proteins involved in DNA repair, thereby causing repair of DNA damage to become less efficient. We previously developed a mathematical model of this potentially synergistic relationship and applied it to γ-ray exposure data on the radiation-resistant prokaryote Deinococcus radiodurans. Here, we investigate the effects of radiation quality on these processes by applying the model to data on exposures of D. radiodurans to heavy ions with linear energy transfer (LET) of 18.5–11,300 keV/μm. The model adequately describes these data using three parameters combinations: radiogenic DNA damage induction, repair protein inactivation and cellular repair capacity. Although statistical uncertainties around best-fit parameter estimates are substantial, the behaviors of model parameters are consistent with current knowledge of LET effects: inactivation cross-sections for both DNA and proteins increase with increasing LET; DNA damage yield per unit of radiation dose also increases with LET; protein damage per unit dose tends to decrease with LET; DNA and especially protein damage yields are reduced when cells are irradiated in the dry state. These results suggest that synergism between oxidative stress and DNA damage may play an important role not only during γ-ray exposure, but during high-LET radiation exposure as well.     

Deinococcus radiodurans DR1088 is a novel RecF‐interacting protein that stimulates single‐stranded DNA annealing

RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF‐dr1088 operon, which is highly conserved in the Deinococcus‐Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two‐hybrid and pull‐down assays. DR1088 exhibited some RecO‐like biochemical properties including single/double‐stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity. However, unlike other recombination proteins, dr1088 is essential for cell viability. These results indicate that DR1088 might play a role in DNA replication and DNA repair processes.     

抗辐射菌Deinococcus radiodurans的多样性

抗辐射菌Deinococcus radiodurans是一种对电离辐射和其他DNA损伤因子具有极强抵抗能力的细菌,是研究DNA损伤与修复的模式生物。综述了国内外在抗辐射菌研究上取得的最新研究成果,从生存环境、对DNA损伤因子的抗性、抗性机理及其损伤修复关联基因等方面报道了抗辐射菌的多样性,并探讨了该细菌高效正确的DNA损伤修复机理的相关研究成果在生命科学、农业、环境修复及医学等领域的应用前景。     

DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm⁻², which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6–4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.     

Preserving Genome Integrity: The DdrA Protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

High cell density production of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> under optimized conditions

Deinococcus radiodurans is a bacterium being investigated for mechanisms of extreme radiation resistance and for bioremediation of environmental radioactive waste sites. In both fundamental and applied research settings, methods for large-scale production of D. radiodurans are needed. In this study, a systematic investigation was carried out to optimize D. radiodurans production at the 20-L fermentor scale. In defined medium, the phosphate buffer typically used was found to be inhibitory to D. radiodurans growth, and caused cell aggregation. Substitution of HEPES and MOPS buffers for phosphate buffer improved D. radiodurans growth characteristics. Several antifoaming agents were investigated to support large-scale production with submerged aeration, and the defoamer KFO 673 was chosen based on its ability to prevent foaming without affecting D. radiodurans growth. The conventional undefined rich medium tryptone/glucose/yeast extract (TGY) maximally supported D. radiodurans growth to an OD₆₀₀ of 10. Using a ‘design of experiments’ approach, we found glucose, Mg and Mn to be critical in supporting high-density growth of D. radiodurans. The optimal pH and temperature for D. radiodurans growth in large-scale preparations were 7.0 and 37°C, respectively. Growth was carried out in a 20-L fermentor using the newly developed media under the optimal conditions. With addition of 10 g/L glucose, 0.5 g/L MgSO₄ · 7H₂O, 5 μM MnCl₂ into TGY media, an OD₆₀₀ of 40 was achieved.     

Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations

Background  

DNA ligases are required for DNA strand joining in all forms of cellular life. NAD⁺-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD⁺-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx.volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx.volcanii ATP-dependent DNA ligase protein LigA.