PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans

Deinococcus radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, particularly, ionizing radiation. Available evidence indicates that efficient repair of DNA damage and protection of the chromosomal structure are mainly responsible for the radioresistance. Little is known about the biochemical basis for this phenomenon. We have identified a unique gene, pprI, as a general switch for downstream DNA repair and protection pathways, from a natural mutant, in which pprI is disrupted by a transposon. Complete functional disruption of the gene in wild-type leads to dramatic sensitivity to ionizing radiation. Radioresistance of the disruptant could be fully restored by complementation with pprI. In response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection pathways in response to radiation stress.     

An exonuclease I-sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance

Deinococcus radiodurans R1 recovering from acute dose of gamma radiation shows a biphasic mechanism of DNA double-strand break repair. The possible involvement of microsequence homology-dependent, or non-homologous end joining type mechanisms during initial period followed by RecA-dependent homologous recombination pathways has been suggested for the reconstruction of complete genomes in this microbe. We have exploited the known roles of exonuclease I in DNA recombination to elucidate the nature of recombination involved in DNA double-strand break repair during post-irradiation recovery of D. radiodurans. Transgenic Deinococcus cells expressing exonuclease I functions of Escherichia coli showed significant reduction in gamma radiation radioresistance, while the resistance to far-UV and hydrogen peroxide remained unaffected. The overexpression of E. coli exonuclease I in Deinococcus inhibited DNA double-strand break repair. Such cells exhibited normal post-irradiation expression kinetics of RecA, PprA and single-stranded DNA-binding proteins but lacked the divalent cation manganese [(Mn(II)]-dependent protection from gamma radiation. The results strongly suggest that 3' (rho) 5' single-stranded DNA ends constitute an important component in recombination pathway involved in DNA double-strand break repair and that absence of sbcB from deinococcal genome may significantly aid its extreme radioresistance phenotype.     

抗辐射菌中DNA损伤修复主要基因群的研究进展

抗辐射红色球菌对电离辐射具有很高的放射线抵抗性,该菌具有惊人的DNA的二条链切断的修复能力,由辐射等引起的切断损伤DNA在几至十几小时内能高效正确地进行完全修复。在对切断的双链DNA进行修复时,除了大肠杆菌等生物在切断的双链DNA修复时出现的蛋白质以外,还有该菌所特有的修复蛋白质也参与修复。本文对该菌所特有的DNA二条链的切断损伤修复的主要基因及其相互作用进行了简要介绍。     

耐辐射球菌pprI在大肠杆菌中表达增强细胞抗氧化能力的研究

将耐辐射球菌(Deinococcus radiodurans)与DNA修复有关的开关基因—pprI通过穿梭质粒pRADZ3导入大肠杆菌TG1中,使其在正常培养条件下(不需诱导剂)表达PprI蛋白,并通过Western blot证实该基因在TG1中可稳定表达。与转化了空白质粒pRADZ3 TG1对照,观察了改造后的两种大肠杆菌在有H2O2氧化压力下的存活率和大肠杆菌中两种过氧化氢酶（KatE, KatG）的活性表达差异。结果表明,无论在指数生长期还是稳定生长期,能表达PprI蛋白的大肠杆菌比对照的存活率要高出10％左右;非变性电泳结果表明,耐辐射球菌pprI 在大肠杆菌中的表达使得KatE活性在指数生长期与稳定生长期分别增加1.5～2倍和2.5～3倍。证明耐辐射球菌pprI 在大肠杆菌中的表达能够增强细胞抗氧化能力。     

RecBC enzyme overproduction affects UV and gamma radiation survival of Deinococcus radiodurans

Deinococcus radiodurans recovering from the effect of acute dose of gamma (gamma) radiation shows a biphasic mechanism of DNA double strands breaks repair that involves an efficient homologous recombination. However, it shows higher sensitivity to near-UV (NUV) than Escherichia coli and lacks RecBC, a DNA strand break (DSB) repair enzyme in some bacteria. Recombinant Deinococcus expressing the recBC genes of E. coli showed nearly three-fold improvements in near-UV tolerance and nearly 2 log cycle reductions in wild type gamma radiation resistance. RecBC over expression effect on radiation response of D. radiodurans was independent of indigenous RecD. Loss of gamma radiation tolerance was attributed to the enhanced rate of in vivo degradation of radiation damaged DNA and delayed kinetics of DSB repair during post-irradiation recovery. RecBC expressing cells of Deinococcus showed wild type response to Far-UV. These results suggest that the overproduction of RecBC competes with the indigenous mechanism of gamma radiation damaged DNA repair while it supports near-UV tolerance in D. radiodurans.     

Identification of a DNA processing complex from Deinococcus radiodurans

An efficient DNA strand break repair contributes to the radioresistance of Deinococcus radiodurans, which harbors the DNA repair pathways nearly identical to Escherichia coli. The molecular mechanisms of these proteins functioning in 2 diverse classes of bacteria seem to be different. The macromolecular interactions and formation of multiprotein complexes in vivo have gained significant importance in explaining the mechanism of the complex cellular processes. Here, we report the identification of a novel DNA metabolic protein complex from D. radiodurans. A similar complex has, however, not been found in E. coli. Mass spectrometric analysis showed the presence of a few known DNA repair proteins, molecular chaperones, and a large number of uncharacterized proteins from D. radiodurans R1. Biochemical and immunoblotting results indicated the presence of the protein promoting DNA repair A, DNA polymerase, Mg2+, and (or) Mn2+ -dependent 5'-->3' exonuclease activity along with protein kinase activity and phosphoproteins. DNA ligase activity was completely dependent upon the ATP requirement, as no ligase activity was seen in the presence of NAD as a cofactor. These results suggest the molecular interactions of the known DNA repair proteins with uncharacterized proteins in the macromolecular complex and the regulation of DNA degradation with the involvement of ATP and protein kinase functions.     

Mechanistic analysis of the contributions of DNA and protein damage to radiation-induced cell death

Protein oxidation can contribute to radiation-induced cell death by two mechanisms: (1) by reducing the fidelity of DNA repair, and (2) by decreasing cell viability directly. Previously, we explored the first mechanism by developing a mathematical model and applying it to data on Deinococcus radiodurans . Here we extend the model to both mechanisms, and analyze a recently published data set of protein carbonylation and cell survival in D. radiodurans and Escherichia coli exposed to gamma and ultraviolet radiation. Our results suggest that similar cell survival curves can be produced by very different mechanisms. For example, wild-type E. coli and DNA double-strand break (DSB) repair-deficient recA- D. radiodurans succumb to radiation doses of similar magnitude, but for different reasons: wild-type E. coli proteins are easily oxidized, causing cell death even at low levels of DNA damage, whereas proteins in recA- D. radiodurans are well protected from oxidation, but DSBs are not repaired correctly even when most proteins are intact. Radioresistant E. coli mutants survive higher radiation doses than the wild-type because of superior protection of cellular proteins from radiogenic oxidation. In contrast, wild-type D. radiodurans is much more radioresistant than the recA- mutant because of superior DSB repair, whereas protein protection in both strains is similar. With further development, the modeling approach presented here can also quantify the causes of radiation-induced cell death in other organisms. Enhanced understanding of these causes can stimulate research on novel radioprotection strategies.     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌(Deinococcus radiodurans)recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E.coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D.radiodurans的recA基因在E.coli细胞中的表达能够完全补偿recA缺损E.coli辐射抗性能力。     

Gamma radiation-induced proteome of Deinococcus radiodurans primarily targets DNA repair and oxidative stress alleviation

The extraordinary radioresistance of Deinococcus radiodurans primarily originates from its efficient DNA repair ability. The kinetics of proteomic changes induced by a 6-kGy dose of gamma irradiation was mapped during the post-irradiation growth arrest phase by two-dimensional protein electrophoresis coupled with mass spectrometry. The results revealed that at least 37 proteins displayed either enhanced or de novo expression in the first 1 h of post-irradiation recovery. All of the radiation-responsive proteins were identified, and they belonged to the major functional categories of DNA repair, oxidative stress alleviation, and protein translation/folding. The dynamics of radiation-responsive protein levels throughout the growth arrest phase demonstrated (i) sequential up-regulation and processing of DNA repair proteins such as single-stranded DNA-binding protein (Ssb), DNA damage response protein A (DdrA), DNA damage response protein B (DdrB), pleiotropic protein promoting DNA repair (PprA), and recombinase A (RecA) substantiating stepwise genome restitution by different DNA repair pathways and (ii) concurrent early up-regulation of proteins involved in both DNA repair and oxidative stress alleviation. Among DNA repair proteins, Ssb was found to be the first and most abundant radiation-induced protein only to be followed by alternate Ssb, DdrB, indicating aggressive protection of single strand DNA fragments as the first line of defense by D. radiodurans, thereby preserving genetic information following radiation stress. The implications of both qualitative or quantitative and sequential or co-induction of radiation-responsive proteins for envisaged DNA repair mechanism in D. radiodurans are discussed.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

Effect of a recD mutation on DNA damage resistance and transformation in Deinococcus radiodurans

The bacterium Deinococcus radiodurans is resistant to extremely high levels of DNA-damaging agents such as UV light, ionizing radiation, and chemicals such as hydrogen peroxide and mitomycin C. The organism is able to repair large numbers of double-strand breaks caused by ionizing radiation, in spite of the lack of the RecBCD enzyme, which is essential for double-strand DNA break repair in Escherichia coli and many other bacteria. The D. radiodurans genome sequence indicates that the organism lacks recB and recC genes, but there is a gene encoding a protein with significant similarity to the RecD protein of E. coli and other bacteria. We have generated D. radiodurans strains with a disruption or deletion of the recD gene. The recD mutants are more sensitive than wild-type cells to irradiation with gamma rays and UV light and to treatment with hydrogen peroxide, but they are not sensitive to treatment with mitomycin C and methyl methanesulfonate. The recD mutants also show greater efficiency of transformation by exogenous homologous DNA. These results are the first indication that the D. radiodurans RecD protein has a role in DNA damage repair and/or homologous recombination in the organism.     

Evidence against enhancement of the radioresistance of Escherichia coli by cloned Deinococcus radiodurans DNA

Deinococcus radiodurans genomic DNA, introduced to Escherichia coli in cloning vectors, has been reported to produce radioresistant E. coli that can be selected by gamma irradiation. In this report prior results are reassessed experimentally, and additional studies are presented. Results to date suggest that the acquired radioresistance of E. coli selected by gamma irradiation does not stem from expression of stable plasmid-encoded D. radiodurans sequences, and that acquired radioresistance is not readily transmitted to naive (unirradiated) E. coli by transformation of plasmid recovered from the radioresistant isolates. Several interpretations are discussed.     

DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea<Emphasis Type="Italic"> Pyrococcus abyssi</Emphasis> and<Emphasis Type="Italic"> P. furiosus</Emphasis>

Hyperthermophilic archaea of the genus Pyrococcus are resistant to gamma radiation, suggesting that efficient mechanisms for DNA repair exist in these organisms. To determine whether protective mechanisms might also be implicated in this radioresistance, we have estimated the linear density of DNA double-stranded breaks caused by gamma irradiation in the genomic DNA of two Pyrococcus species, using Escherichia coli and the radioresistant bacterium Deinococcus radiodurans as controls. The linear density of double-stranded breaks was essentially the same in all four microorganisms when irradiation was carried under similar anaerobic conditions, indicating that no specific DNA protection mechanisms exist in Pyrococcus species. Using one- and two-dimensional gel electrophoresis we compared the protein patterns from Pyrococcus abyssi and P. furiosus cells that had or had not been exposed to gamma rays. We did not detect any significant protein induction following DNA damage in either species.     

Catalase and superoxide dismutase activity in isogenous bacterial strains with different degrees of radioresistance

Superoxide dismutase and catalase activity has been studied in isogenous strains of various radioresistance bacteria. In mutants Micrococcus radiodurans having defects in the systems of DNA repair the superoxide dismutase activity is lower than in cells of wild type. The changes of catalase and superoxide dismutase activity have not been revealed in investigated strains Escherichia coli differing in radioresistance. It has been concluded that the survival of bacteria exposed to ionizing radiation is determined by the effectiveness of DNA repair systems realiability of which depends on the catalase and superoxide dismutase activity.     

Expression of recA in Deinococcus radiodurans.

Deinococcus (formerly Micrococcus) radiodurans is remarkable for its extraordinary resistance to ionizing and UV irradiation and many other agents that damage DNA. This organism can repair > 100 double-strand breaks per chromosome induced by ionizing radiation without lethality or mutagenesis. We have previously observed that expression of D. radiodurans recA in Escherichia coli appears lethal. We now find that the RecA protein of D. radiodurans is ot detectable in D. radiodurans except in the setting of DNA damage and that termination of its synthesis is associated with the onset of deinococcal growth. The synthesis of Shigella flexneri RecA (protein sequence identical to that of E. coli RecA) in recA-defective D. radiodurans is described. Despite a large accumulation of the S. flexneri RecA in D. radiodurans, there is no complementation of any D. radiodurans recA phenotype, including DNA damage sensitivity, inhibition of natural transformation, or inability to support a plasmid that requires RecA for replication. To ensure that the cloned S. flexneri recA gene was not inactivated, it was rescued from D. radiodurans and was shown to function normally in E. coli. We conclude that neither D. radiodurans nor S. flexneri RecA is functional in the other species, nor are the kinetics of induction and suppression similar to each other, indicating a difference between these two proteins in their modes of action.     

Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli

The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli.     

Deinococcus radiodurans: what belongs to the survival kit?

Deinococcus radiodurans, one of the most radioresistant organisms known to date, is able to repair efficiently hundreds of DNA double- and single-strand breaks as well as other types of DNA damages promoted by ionizing or ultraviolet radiation. We review recent discoveries concerning several aspects of radioresistance and survival under high genotoxic stress. We discuss different hypotheses and possibilities that have been suggested to contribute to radioresistance and propose that D. radiodurans combines a variety of physiological tools that are tightly coordinated. A complex network of regulatory proteins may be discovered in the near future that might allow further understanding of radioresistance.