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.     

Interchromosomal recombination in the extremely radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans and other members of the genus Deinococcus are remarkable for their extreme resistance to ionizing radiation and many other agents that damage DNA. We have recently shown that recombinational processes participate in interplasmidic repair following in vivo irradiation. We now present direct studies on interchromosomal recombination among chromosomes irradiated in vivo during stationary phase (four chromosomes per cell). Following an exposure to 1.75 Mrad (the dose required to achieve a survival of 37%, which degrades the cells' four chromosomes into about 500 fragments), we determined that there may be as many as 175 crossovers per chromosome (700 crossovers per nucleoid) undergoing repair. In addition, these studies suggest that many of the crossovers occurring during repair are nonreciprocal.     

Radiation response mechanisms of the extremely radioresistant bacterium Deinococcus radiodurans.

Effect of microgravity on recovery of bacterial cells from radiation damage was examined in IML-2, S/MM-4 and S/MM-9 experiments using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under the space environment. No difference was observed between the survivals of radiosensitive mutant rec30 cells incubated in space and on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, product of a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance in repair-deficient mutant, KH311, was enhanced under microgravity compared with ground controls. Recent investigation in vitro showed that PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalyzed by DNA ligases. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining (NHEJ) repair mechanism in which PprA plays a critical role.     

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.     

Genome sequence and transcriptome of Sorbus pohuashanensis provide insights into population evolution and leaf sunburn response

Sorbus pohuashanensis (Hance) Hedl. is a potential horticulture and medicinal plant, but its genomic and genetic backgrounds remain unknown. Here, we sequence and assemble the S. pohuashanensis reference genome using PacBio long reads. Based on the new reference genome, we resequence a core collection of 22 Sorbus spp. samples, which are divided into 2 groups (G1 and G2) based on phylogenetic and PCA analyses. These phylogenetic clusters are highly consistent with their classification based on leaf shape. Natural hybridization between the G1 and G2 groups is evidenced by a sample (R21) with a highly heterozygous genotype. Nucleotide diversity (π) analysis shows that G1 has a higher diversity than G2 and that G2 originated from G1. During the evolution process, the gene families involved in photosynthesis pathways expanded and the gene families involved in energy consumption contracted. RNA-seq data suggests that flavonoid biosynthesis and heat-shock protein (HSP)-heat-shock factor (HSF) pathways play important roles in protection against sunburn. This study provides new insights into the evolution of Sorbus spp. genomes. In addition, the genomic resources, and the identified genetic variations, especially those related to stress resistance, will help future efforts to produce and breed Sorbus spp.     

Analysis of codon usage pattern in the radioresistant bacterium Deinococcus radiodurans

The main factors shaping codon usage bias in the Deinococcus radiodurans genome were reported. Correspondence analysis (COA) was carried out to analyze synonymous codon usage bias. The results showed that the main trend was strongly correlated with gene expression level assessed by the "Codon Adaptation Index" (CAI) values, a result that was confirmed by the distribution of genes along the first axis. The results of correlation analysis, variance analysis and neutrality plot indicated that gene nucleotide composition was clearly contributed to codon bias. CDS length was also key factor in dictating codon usage variation. A general tendency of more biased codon usage of genes with longer CDS length to higher expression level was found. Further, the hydrophobicity of each protein also played a role in shaping codon usage in this organism, which could be confirmed by the significant correlation between the positions of genes placed on the first axis and the hydrophobicity values (r=-0.100, P<0.01). In summary, gene expression level played a crucial role, nucleotide mutational bias, CDS length and the hydrophobicity of each protein just in a minor way in shaping the codon usage pattern of D. radiodurans. Notably, 19 codons firstly defined as "optimal codons" may provide useful clues for molecular genetic engineering and evolutionary studying.     

Identification and disruption analysis of the recN gene in the extremely radioresistant bacterium Deinococcus radiodurans.

We isolated a radiosensitive mutant strain, KR4128, from a wild-type strain of Deinococcus radiodurans, which is known as a extremely radioresistant bacterium. The gene that restore the defect of the mutant in DNA repair was cloned, and it turned out to be the homolog of the recN gene of Escherichia coli. The recN gene encoded a protein of 58 kDa, and, in its N-terminal region, a potential ATP binding domain was conserved as expected for a prokaryotic RecN protein. An analysis of sequence of the mutant recN gene revealed a G:C to T:A transversion near the 3' end of the coding region. This alteration causes an ochre mutation, and results in the truncation of 47 amino acids from the C-terminal region of the RecN protein. The null mutant of recN gene was constructed by insertional mutagenesis, and it showed substantial sensitivities to various types of DNA damaging agents, indicating that a single defect in the recN gene can directly affect the DNA damage resistant phenotype in D. radiodurans. The recN locus of KR4128 was also disrupted and the disruptant indicated the sensitivity that was indistinguishable from its progenitor. The result indicate that the transversion in the recN gene of KR4128 cells causes a complete loss of function of the RecN protein and thus the C-terminal region of the RecN protein includes domain essential to its function.     

Interplasmidic recombination following irradiation of the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of the eubacterial family Deinococcaceae are extremely resistant to ionizing radiation and many other agents that damage DNA. For example, after irradiation, D. radiodurans can repair > 100 DNA double-strand breaks per chromosome without lethality or mutagenesis, while most other organisms can survive no more than 2 or 3 double-strand breaks. The unusual resistance of D. radiodurans is recA dependent, but the repair pathway(s) is not understood. Recently, we described how a plasmid present in D. radiodurans (plasmid copy number, approximately 6 per cell; chromosome copy number, approximately 4 per cell) during high-dose irradiation undergoes extreme damage like the chromosome and is retained by the cell without selection and fully repaired with the same efficiency as the chromosome. In the current work, we have investigated the repair of two similar plasmids within the same cell. These two plasmids were designed to provide both restriction fragment polymorphisms and a drug selection indicator of recombination. This study presents a novel system of analysis of in vivo damage and recombinational repair, exploiting the unique ability of D. radiodurans to survive extraordinarily high levels of DNA damage. We report that homologous recombination among plasmids following irradiation is extensive. For example, 2% of Tcs plasmids become Tcr as a result of productive recombination within a 929-bp region of the plasmids after repair. Our results suggest that each plasmid may participate in as many as 6.7 recombinational events during repair, a value that extrapolates to > 700 events per chromosome undergoing repair simultaneously. These results indicate that the study of plasmid recombination within D. radiodurans may serve as an accurate model system for simultaneously occurring repair in the chromosome.     

Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.     

Characterization of the DdrD protein from the extremely radioresistant bacterium Deinococcus radiodurans

Extremophiles - Here, we report the in vitro and in vivo characterization of the DdrD protein from the extraordinary stress-resistant bacterium, D. radiodurans. DdrD is one of the most highly...     

RecX is involved in antioxidant mechanisms of the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans shows remarkable resistance to reactive oxygen species (ROS), generated by irradiation. Disruption of recX (dr1310) in D. radiodurans using targeted mutagenesis method enhanced its ROS scavenging activity, and recX overexpression in this bacterium repressed its antioxidant activity significantly. Further analyses on catalase and superoxide dismutase, two important antioxidant proteins in cells, showed that RecX could repress the induction of antioxidant enzymes, revealing that it negatively regulates the ROS scavenging activity in D. radiodurans.     

耐辐射球菌基因DRB0099缺失突变株的构建及逆境分析

耐辐射球菌(Deinococcus radiodurans R1)有着极强的辐射抗性.研究其抗辐射的机理对于处理放射性废料有着潜在的应用价值.在耐辐射球菌的基因组中,许多序列的功能未知.其中DRB0099尤为引人注意.将DRB0099缺失突变构建该基因的突变株.对野生型和突变体进行比较后发现,在正常生长条件下的前期阶段(0～16 h),突变体生长速度比野生型慢.16 h以后,野生型逐渐进入稳定生长期.这时,突变株的生长速度高于野生型.但是,野生型的浓度一直高于突变株.表明在DRB0099被删除后,耐辐射球菌的生长可能受到了阻滞.在紫外线照射的条件下,尽管野生型随着照射剂量的增加,存活率越来越低,但是要比突变体高许多.野生型具有比突变体更强的修复DNA双链断裂的能力.DRB0099可能直接参与了对DNA的修复.突变体对H2O2的敏感程度高于野生型,表明野生型耐辐射球菌在对抗活性氧保护其蛋白质、DNA或者DNA修复方面具有比突变体更强的功能.在低浓度H2O2处理条件下,尽管野生型和突变体的存活率都出现下降趋势,但二者的差值并不大.随着H2O2剂量的增加,二者的差值越来越大.表明随着活性氧浓度的增加,蛋白质和DNA损伤的数量增加,失去DRB0099基因功能的突变体比野生型更容易受到损伤.在紫外线照射处理或者H2O2处理条件下,DRB0099能够保护蛋白质和DNA.     

Crystal structure of RecA from Deinococcus radiodurans: insights into the structural basis of extreme radioresistance

The resistance of Deinococcus radiodurans (Dr) to extreme doses of ionizing radiation depends on its highly efficient capacity to repair dsDNA breaks. Dr RecA, the key protein in the repair of dsDNA breaks by homologous recombination, promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA. In order to gain insight into the remarkable repair capacity of Dr and the novel mechanistic features of its RecA protein, we have determined its X-ray crystal structure in complex with ATPgammaS at 2.5A resolution. Like RecA from Escherichia coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67 A. Although the overall fold of Dr RecA is similar to E.coli RecA, there is a large reorientation of the C-terminal domain, which in E.coli RecA has a site for binding dsDNA. Compared to E.coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential. Unique amino acid residues in Dr RecA cluster around a flexible beta-hairpin that has also been implicated in DNA binding.     

Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.

Escherichia coli drug resistance plasmids were introduced into Deinococcus radiodurans by cloning D. radiodurans DNA into the plasmids prior to transformation. The plasmids were integrated into the chromosome of the transformants and flanked by a direct repeat of the cloned D. radiodurans segment. The plasmid and one copy of the flanking chromosomal segment constituted a unit ("amplification unit") which was found repeated in tandem at the site of chromosomal integration. Up to 50 copies of the amplification unit were present per chromosome, accounting for approximately 10% of the genomic DNA. Circular forms of the amplification unit were also present in D. radiodurans transformants. These circles were introduced into E. coli, where they replicated as plasmids. The drug resistance determinants which have been introduced into D. radiodurans in this fashion are cat (from Tn9) and aphA (from Tn903). Transformation of D. radiodurans to drug resistance was efficient when the donor DNA was from D. radiodurans or E. coli, but was greatly reduced when the donor DNA was linearized with restriction enzymes prior to transformation. In the course of the study, a plasmid, pS16, was discovered in D. radiodurans R1, establishing that all Deinococcus strains so far examined contain plasmids.     

Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.     

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.     

Global whole-cell FTICR mass spectrometric proteomics analysis of the heat shock response in the radioresistant bacterium Deinococcus radiodurans

The results of previous studies indicated that D. radiodurans mounts a regulated protective response to heat shock, and that expression of more than 130 genes, including classical chaperones such as the groESL and dnaKJ operons and proteases such as clpB are induced in response to elevated temperature. In addition, previous qualitative whole-cell mass spectrometric studies conducted under heat shock conditions indicated global changes in the D. radiodurans proteome. To enable the discovery of novel heat shock inducible proteins as well as gain greater biological insight into the classical heat shock response at the protein level, we undertook the global whole-cell FTICR mass spectrometric proteomics study reported here. We have greatly increased the power of this approach by conducting a large number of replicate experiments in addition to taking a semiquantitative approach to data analysis, finding good reproducibility between replicates. Through this analysis, we have identified with high confidence a core set of classical heat shock proteins whose expression increases dramatically and reproducibly in response to elevated temperature. In addition, we have found that the heat shock proteome includes a large number of induced proteins that have not been identified previously as heat responsive, and have therefore been designated as candidate responders. Finally, our results are consistent with the hypothesis that elevated temperature stress could lead to cross-protection against other related stresses.