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 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.     

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。     

Additive effects of SbcCD and PolX deficiencies in the in vivo repair of DNA double-strand breaks in Deinococcus radiodurans

Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following gamma-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3'-to-5' exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli. We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant DeltasbcCD DeltapolX(Dr) (where Dr indicates D. radiodurans) bacteria are much more sensitive to gamma-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair.     

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.     

DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR 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.     

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.     

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.     

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.     

Function and biochemical characterization of RecJ in Deinococcus radiodurans

The single-stranded DNA-specific nuclease RecJ is found in most bacteria where it is involved in the RecFOR double-stranded break (DSBs) repair pathway. DSBs repair mainly occurs via the RecFOR pathway in Deinococcus radiodurans, a well-known radiation-resistant bacterium. A recJ null mutant was constructed to investigate the role of recJ in D. radiodurans. recJ inactivation caused growth defects and sensitivity to high temperatures. However, the radiation resistance of the recJ mutant was only moderately decreased. The full-length D. radiodurans RecJ (DrRecJ) protein was expressed and purified to further characterize its biochemical properties. DrRecJ possessed a Mn(2+) concentration-dependent nuclease activity where the optimal Mn(2+) concentration was 0.1mM. DrRecJ had a similar activity profile after adding 10mM Mg(2+) to reactions with different Mn(2+) concentrations, indicating that Mn(2+) is a RecJ regulator. Escherichia coli RecJ has no activity on 5' ssDNA tails shorter than 6-nt, but DrRecJ could effectively degrade DNA with a 4-nt 5' ssDNA tail, suggesting that DrRecJ may have a wider range of DNA substrates. Moreover, SSB in D. radiodurans stimulated the DrRecJ exonuclease activity, whereas DdrB inhibited it and provided protection to ssDNA. Overall, our results indicate that recJ is a nonessential gene in D. radiodurans and that the activity of DrRecJ is regulated by Mn(2+) and SSB-DdrB.     

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.     

Investigating Deinococcus radiodurans RecA protein filament formation on double-stranded DNA by a real-time single-molecule approach

With the aid of an efficient, precise, and almost error-free DNA repair system, Deinococcus radiodurans can survive hundreds of double-strand breaks inflicted by high doses of irradiation or desiccation. RecA of D. radiodurans (DrRecA) plays a central role both in the early phase of repair by an extended synthesis-dependent strand annealing process and in the later more general homologous recombination phase. Both roles likely require DrRecA filament formation on duplex DNA. We have developed single-molecule tethered particle motion experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules by observing changes in DNA tether length resulting from RecA binding. We demonstrate that DrRecA nucleation on double-stranded DNA is much faster than that of Escherichia coli RecA protein (EcRecA), but the extension is slower. This combination of attributes would tend to increase the number and decrease the length of DrRecA filaments relative to those of EcRecA, a feature that may reflect the requirement to repair hundreds of genomic double-strand breaks concurrently in irradiated Deinococcus cells.     

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.     

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.     

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.     

Biochemical characterization of two DNA ligases from Deinococcus radiodurans

Two genes encoding a NAD(+)-dependent DNA ligase (LigA) and an ATP-dependent DNA ligase (LigB) were identified in the genome of the extremely radioresistant bacterium, Deinococcus radiodurans (DR). The recombinant enzymes expressed in Escherichia coli, were purified to homogeneity and characterized. The optimal temperature and pH value of the two DNA ligases were 60 ( degrees )C and 7.0, respectively. Their optimal concentration of MgCl(2) was 5mM. Their half-lifes of heat inactivation at 100 ( degrees )C were about 3 min and 5 min, respectively. In addition, the results showed that DRLigB displayed higher activity than DRLigA at stick and blunt ended joining of DNA, indicating that DRLigB is a key DNA ligase of D. radiodurans in DNA recombination and double-strand break repair.     

Dysregulation of IRP1-Mediated Iron Metabolism Causes Gamma Ray-specific Radioresistance in Leukemia Cells

Iron is required for nearly all organisms, playing important roles in oxygen transport and many enzymatic reactions. Excess iron, however, can be cytotoxic. Emerging evidence suggests that radioresistance can be achieved in lower organisms by the protection of proteins, but not DNA, immediately following ionizing radiation (IR) exposure, allowing for improved DNA repair. One potential mechanism for protein protection is controlling and limiting the amount of free iron in cells, as has been demonstrated in the extremophile Deinococcus Radiodurans, reducing the potential for oxidative damage to proteins during exposure to IR. We found that iron regulatory protein 1 (IRP1) expression was markedly reduced in human myeloid leukemia HL60 cells resistant to low linear energy transfer (LET) gamma rays, but not to high LET alpha particles. Stable knockdown of IRP1 by short-hairpin RNA (shRNA) interference in radiosensitive parental cells led to radioresistance to low LET IR, reduced intracellular Fenton chemistry, reduced protein oxidation, and more rapid DNA double-strand break (DSB) repair. The mechanism of radioresistance appeared to be related to attenuated free radical-mediated cell death. Control of intracellular iron by IRPs may be a novel radioresistance mechanism in mammalian cells.