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.     

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.     

耐辐射异常球菌抗辐射机理的研究新进展

报道了自1956年Anderson发现耐辐射异常球菌(Deinococcusradiodurans)以来,国外在其生理生化和遗传学特性、特殊的细胞膜结构、各种诱变因素所致的DNA损伤与其高效的修复机制和生物化学、分子生物学应用于该菌的研究新进展。对该菌的研究在辐射生物学与医学上具有特殊的意义,因此,我国的辐射生物学、微生物学和医学研究人员应尽快开展这方面的研究。     

Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans.

Deinococcus radiodurans and other species of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. Two different DNA damage-sensitive strains generated by chemical mutagenesis were found to be defective in a gene that has extended DNA and protein sequence homology with polA of Escherichia coli. Both mutant strains lacked DNA polymerase, as measured in activity gels. Transformation of this gene from wild-type D. radiodurans restored to the mutants both polymerase activity and DNA damage resistance. A technique for targeted insertional mutagenesis in D. radiodurans is presented. This technique was employed to construct a pol mutant isogenic with the wild type (the first example of targeted mutagenesis in this eubacterial family). This insertional mutant lacked DNA polymerase activity and was even more sensitive to DNA damage than the mutants derived by chemical mutagenesis. In the case of ionizing radiation, the survival of the wild type after receiving 1 Mrad was 100% while survival of the insertional mutant extrapolated to 10(-24). These results demonstrate that the gene described here encodes a DNA polymerase and that defects in this pol gene cause a dramatic loss of resistance of D. radiodurans to DNA damage.     

Expression of Deinococcus radiodurans PprI enhances the radioresistance of Escherichia coli

PprI, a newly identified gene switch responsible for extreme radioresistance of Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair and protection pathways in response to radiation stress [Biochem. Biophy. Res. Commun. 306 (2003) 354]. To evaluate whether PprI also functions in the radioresistance in other organisms, D. radiodurans PprI protein (Deira-PprI) was expressed in Escherichia coli. The complemented E. coli strain showed an increase of approximately 1.6-fold radioresistance with a high dose of gamma irradiation. Immunoblotting assays showed that the expression of Deira-PprI in E. coli resulted in a significant increase in RecA protein expression following high dose ionizing radiation. The expression of Deira-PprI protein also significantly enhanced the scavenging ability of free radicals by inducing the enzymatic activity of KatG. These results indicate that exogenous expression of Deira-PprI promotes DNA repair and protection pathways and enhances the radioresistance of E. coli.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

Structure of a novel phosphoglycolipid from Deinococcus radiodurans

The chemical structure of a major phosphoglycolipid from Deinococcus radiodurans has been shown to be 2'-O-(1,2-diacyl-sn-glycero-3-phospho)-3'-O-(alpha-galactosyl)-N-D-gl yceroyl alkylamine. By infrared spectroscopy, the lipid was shown to contain both carbonyl ester and amide linkages. Chemical analysis demonstrated a molar ratio of fatty acid, carbohydrate, and phosphorus of 2:1:1. The lipid was shown to contain an sn-3-phosphatidic acid backbone by digestion with phospholipase A2. Phosphodiester bond cleavage of the lipid with hydrofluoric acid liberated a component which contained galactose, glyceric acid, and alkylamines. Using NMR and permethylation/hydrolysis procedures, galactose was shown to be linked alpha-glycosidically to the 3-O-position of glyceric acid.     

Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.     

Structure of a novel glucosamine-containing phosphoglycolipid from Deinococcus radiodurans

The structure of a major novel lipid from Deinococcus radiodurans has been determined to be 2'-O-(1,2-diacyl-sn-glycero-3-phospho)-3'-O-(alpha-N-acetylglucosaminyl) -N- glyceroyl alkylamine. The lipid was shown to contain a phosphatidic acid backbone by digestion with phospholipase A2 and by hydrolysis with hydrofluoric acid. Using a combination of chemical and NMR spectroscopic techniques, the structure of this lipid was elucidated and compared with that of a similar phosphoglycolipid reported earlier (Anderson, R., and Hansen, K. (1985) J. Biol. Chem. 260, 12219-12223) in which galactose was found in place of N-acetylglucosamine. The fatty acid compositions of the two lipids were similar.     

PprA: a protein implicated in radioresistance of Deinococcus radiodurans stimulates catalase activity in Escherichia coli

PprA: a pleiotropic protein promoting DNA repair, role in radiation resistance of Deinococcus radiodurans was demonstrated. In this study, the effect of radiation and oxidative stress on transgenic Escherichia coli expressing pprA has been studied. The pprA gene from D. radiodurans KR1 was cloned and expressed in E. coli. Transgenic E. coli cells expressing PprA showed twofold to threefold higher tolerance to hydrogen peroxide as compared to control. The 2.8-fold in vivo stimulation of catalase activity largely contributed by KatE was observed as compared to nonrecombinant control. Furthermore, the purified PprA could stimulate the E. coli catalase activity by 1.7-fold in solution. The effect of PprA on catalase activity observed both in vivo and in vitro was reverted to normal levels in the presence of PprA antibodies. The results suggest that enhanced oxidative stress tolerance in E. coli expressing PprA was due to the PprA stimulation of catalase activity, perhaps through the interaction of these proteins.     

RecO is essential for DNA damage repair in Deinococcus radiodurans

Here we present direct evidence for the vital role of RecO in Deinococcus radiodurans's radioresistance. A recO null mutant was constructed using a deletion replacement method. The mutant exhibited a growth defect and extreme sensitivity to irradiation with gamma rays and UV light. These results suggest that DNA repair in this organism occurs mainly via the RecF pathway.     

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.     

PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation

The extraordinary radiation resistance of Deinococcus radiodurans results from the efficient capacity of the bacterium to repair DNA double-strand breaks. By analysing the DNA damage repair-deficient mutant, KH311, a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance was identified. Investigations in vitro showed that the gene product of pprA (PprA) preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATP-dependent and NAD-dependent DNA ligases. These results suggest that D. radiodurans has a radiation-induced non-homologous end-joining repair mechanism in which PprA plays a critical role.     

Phosphatidylglyceroylalkylamine, a novel phosphoglycolipid precursor in Deinococcus radiodurans.

We report here the structure of a previously uncharacterized phospholipid in the radiation-resistant bacterium Deinococcus radiodurans. This phospholipid, designated lipid 4, was shown by chemical analysis, HF hydrolysis, and nuclear magnetic resonance spectroscopy to be phosphatidylglyceroylalkylamine. Lipid 4 thus contains the unusual lipid constituents glyceric acid and alkylamines, which have previously been identified in two complex phosphoglycolipids from this organism. By [32P]phosphate pulse-chase labeling techniques, lipid 4 was shown to be the precursor of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminylphosphatidylglyceroylalkylamine. While phosphatidylglyceroylalkylamine is rapidly biosynthesized from Pi, its subsequent glycosylation occurs much more slowly. Therefore, we conclude that the final glycosylation step is the rate-limiting event in the biosynthesis of the complex phosphoglycolipids alpha-galactosyl- and alpha-N-acetylglucosaminyl-phosphatidylglyceroylalkylamine.