Lesion bypass activity of DNA polymerase A from the extremely radioresistant organism Deinococcus radiodurans

The bacterium Deinococcus radiodurans survives extremely high exposure to ionizing radiation and extended periods of desiccation. Radiation at the survival doses is known to cause numerous DNA damage, such as hundreds of double strand breaks and single strand breaks, as well as damage of the nucleobases. The mechanisms of D. radiodurans to survive the depicted threats are still only beginning to be understood. DNA polymerase A (PolA) has been shown to be crucially involved in irradiation resistance mechanisms of D. radiodurans. We expressed and characterized the DNA polymerase domain of PolA for the first time in vitro. The obtained enzyme is able to efficiently catalyze DNA-dependent DNA synthesis requiring Mg(II) as divalent metal ion. Additionally, strand displacement synthesis of the DNA polymerase, which is required in several repair processes, could be detected. We further found that DNA polymerase function of PolA is modulated by the presence of Mn(II). Whereas proceeding DNA synthesis of PolA was blocked by certain DNA damage that occurs through radiation of DNA, bypass was facilitated by Mn(II). Our results suggest an enzyme modulator function of Mn(II). These observations parallel reports that D. radiodurans accumulates intracellular Mn(II) in cases of irradiation and that the level of irradiation protection correlates with Mn(II) concentrations.     

耐辐射球菌DNA双链断裂修复机制研究新进展

耐辐射球菌对于电离辐射等DNA损伤剂具有极强的抗性,能够将同一个基因组中同时产生的高达100个以上的DNA双链断裂在数十小时内高效而精准地进行修复,是研究DNA双链断裂修复机制的重要模式生物。同源重组、非同源末端连接和单链退火途径作为3个主要的修复途径参与了耐辐射球菌基因组DNA双链断裂的修复过程。此外,一系列新发现的重要蛋白质,如Ppr I、Ddr B等对于耐辐射球菌基因组的修复过程同样至关重要。根据本实验室和国内外在这一研究领域近年来的报道,以不同的修复途径为线索,综述该菌DNA双链断裂修复机制的最新研究成果。     

Radiation resistance of Deinococcus radiodurans R1 with respect to growth phase

Deinococcus species exhibit an extraordinary ability to withstand ionizing radiation (IR). Most of the studies on radiation resistance have been carried out with exponential phase cells. The studies on radiation resistance of Deinococcus radiodurans R1 with respect to different phases of growth showed that late stationary phase cells of D. radiodurans R1 were fourfold more sensitive to IR and heat as compared with exponential or early stationary phase cells. The increased sensitivity of D. radiodurans R1 to IR in the late stationary phase was not due to a decrease in the intracellular Mn/Fe ratio or an increase in the level of oxidative protein damage. The resistance to IR was restored when late stationary phase cells were incubated for 15 min in fresh medium before irradiation, indicating that replenishment of exhausted nutrients restored the metabolic capability of the cells to repair DNA damage. These observations suggest that stress tolerance mechanisms in D. radiodurans R1 differ from established paradigms.     

Role of RecA in DNA damage repair in Deinococcus radiodurans

It has been shown previously that the RecA protein of Deinococcus radiodurans plays a unique role in the repair of DNA damage in this highly DNA damage-resistant organism. Despite the high level of amino-acid identity, previous work has shown that Escherichia coli RecA does not complement D. radiodurans RecA mutants, further suggesting the uniqueness of D. radiodurans RecA. The work presented here shows that E. coli RecA does in fact provide partial complementation to a D. radiodurans RecA null mutant, suggesting that the RecA protein from D. radiodurans may not be as unique as believed previously.     

Deinococcus radiodurans strain R1 contains N6-methyladenine in its genome

Methylation of DNA is known to be involved in DNA repair mechanisms in bacteria. Deinococcus radiodurans strain R1 on exposure to high radiation undergoes significant DNA damage, which is repaired without mutations. However, the presence of modified nucleotides has not been reported in its genome. We report here the detection of N6-methyladenine in the genome of D. radiodurans strain R1 using immunochemical techniques. This N6-methyladenine is not a part of GATC restriction-modification system. D. radiodurans cell extract also exhibited a DNA adenine methyltransferase activity which was reduced in the early post-irradiation recovery phase.     

Protein oxidation implicated as the primary determinant of bacterial radioresistance

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.     

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.     

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。     

Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation

Although Dienococcus radiodurans is notoriously resistant to far-ultraviolet radiation (FUV; 254 nm), it is highly sensitive to near-ultraviolet radiation (NUV; 300-400 nm), thus demonstrating that the mechanisms of damage (and/or recovery) by the two types of irradiation are different. This observed difference between FUV and NUV effects in D. radiodurans agrees with previous studies with Escherichia coli. Near-ultraviolet radiation produces DNA damage which is presumed to be single-strand breaks (SSB) in the DNA of D. radiodurans. Unique lesions, such as DNA-protein crosslinks could not be demonstrated in this study. Cells that were pre-irradiated with a small dose of NUV were subsequently protected against inactivating doses of NUV. The data presented are consistent with induced DNA repair following NUV damage in D. radiodurans; this is in contrast to FUV damage where DNA repair is constitutive but not induced.     

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.     

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.     

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.     

Physiologic determinants of radiation resistance in Deinococcus radiodurans

Immense volumes of radioactive wastes, which were generated during nuclear weapons production, were disposed of directly in the ground during the Cold War, a period when national security priorities often surmounted concerns over the environment. The bacterium Deinococcus radiodurans is the most radiation-resistant organism known and is currently being engineered for remediation of the toxic metal and organic components of these environmental wastes. Understanding the biotic potential of D. radiodurans and its global physiological integrity in nutritionally restricted radioactive environments is important in development of this organism for in situ bioremediation. We have previously shown that D. radiodurans can grow on rich medium in the presence of continuous radiation (6,000 rads/h) without lethality. In this study we developed a chemically defined minimal medium that can be used to analyze growth of this organism in the presence and in the absence of continuous radiation; whereas cell growth was not affected in the absence of radiation, cells did not grow and were killed in the presence of continuous radiation. Under nutrient-limiting conditions, DNA repair was found to be limited by the metabolic capabilities of D. radiodurans and not by any nutritionally induced defect in genetic repair. The results of our growth studies and analysis of the complete D. radiodurans genomic sequence support the hypothesis that there are several defects in D. radiodurans global metabolic regulation that limit carbon, nitrogen, and DNA metabolism. We identified key nutritional constituents that restore growth of D. radiodurans in nutritionally limiting radioactive environments.     

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.     

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.     

Polar destabilization of DNA duplexes with single-stranded overhangs by the Deinococcus radiodurans SSB protein

The Deinococcus radiodurans SSB protein has an occluded site size of 50 +/- 2 nucleotides on ssDNA but can form a stable complex with a 26-30-nucleotide oligodeoxynucleotide using a subset of its four ssDNA binding domains. Quantitative estimates of D. radiodurans SSB protein in the D. radiodurans cell indicate approximately 2500-3000 dimers/cell, independent of the level of irradiation. At biologically relevant concentrations, when bound at single-strand-double-strand DNA junctions in vitro, D. radiodurans SSB protein has a limited capacity to displace the shorter strand of the duplex, permitting it to bind to single-strand extensions shorter than 26-30 nucleotides. The capacity to displace the shorter strand of the duplex shows a pronounced bias for extensions with a free 3' end. The Escherichia coli SSB protein has a similar but somewhat less robust capacity to displace a DNA strand annealed adjacent to a single-strand extension. These activities are likely to be relevant to the action of bacterial SSB proteins in double-strand break repair, acting at the frayed ends created by ionizing radiation.     

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.     

Deinococcus radiodurans DNA increases the radiation resistance of Escherichia coli

A genomic DNA library of Deinococcus radiodurans DNA has been prepared using the plasmid vector pBR322. The recombinant plasmid was used to transform a more radiation-sensitive organism, Escherichia coli RR1. Following selection of transformed organisms by their ability to grow on ampicillin, radiation-resistant organisms were selected by irradiation with 137Cs gamma radiation. Increased radiation resistance correlates with the presence of a 3-kb fragment of DNA in these cells which is derived from D. radiodurans.     

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.     

The crystal structure of mismatch-specific uracil-DNA glycosylase (MUG) from Deinococcus radiodurans reveals a novel catalytic residue and broad substrate specificity

Deinococcus radiodurans is extremely resistant to the effects of ionizing radiation. The source of the radiation resistance is not known, but an expansion of specific protein families related to stress response and damage control has been observed. DNA repair enzymes are among the expanded protein families in D. radiodurans, and genes encoding five different uracil-DNA glycosylases are identified in the genome. Here we report the three-dimensional structure of the mismatch-specific uracil-DNA glycosylase (MUG) from D. radiodurans (drMUG) to a resolution of 1.75 angstroms. Structural analyses suggest that drMUG possesses a novel catalytic residue, Asp-93. Activity measurements show that drMUG has a modified and broadened substrate specificity compared with Escherichia coli MUG. The importance of Asp-93 for activity was confirmed by structural analysis and abolished activity for the mutant drMUGD93A. Two other microorganisms, Bradyrhizobium japonicum and Rhodopseudomonas palustris, possess genes that encode MUGs with the highest sequence identity to drMUG among all of the bacterial MUGs examined. A phylogenetic analysis indicates that these three MUGs form a new MUG/thymidine-DNA glycosylase subfamily, here called the MUG2 family. We suggest that the novel catalytic residue (Asp-93) has evolved to provide drMUG with broad substrate specificity to increase the DNA repair repertoire of D. radiodurans.