Enzymes involved in DNA ligation and end-healing in the radioresistant bacterium <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Background  

Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of γ-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair.     

Oxidative Stress Resistance in Deinococcus radiodurans

Summary: Deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive DNA damage efficiently and accurately. It is extremely resistant to many DNA-damaging agents, including ionizing radiation and UV radiation (100 to 295 nm), desiccation, and mitomycin C, which induce oxidative damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species. The extreme resilience of D. radiodurans to oxidative stress is imparted synergistically by an efficient protection of proteins against oxidative stress and an efficient DNA repair mechanism, enhanced by functional redundancies in both systems. D. radiodurans assets for the prevention of and recovery from oxidative stress are extensively reviewed here. Radiation- and desiccation-resistant bacteria such as D. radiodurans have substantially lower protein oxidation levels than do sensitive bacteria but have similar yields of DNA double-strand breaks. These findings challenge the concept of DNA as the primary target of radiation toxicity while advancing protein damage, and the protection of proteins against oxidative damage, as a new paradigm of radiation toxicity and survival. The protection of DNA repair and other proteins against oxidative damage is imparted by enzymatic and nonenzymatic antioxidant defense systems dominated by divalent manganese complexes. Given that oxidative stress caused by the accumulation of reactive oxygen species is associated with aging and cancer, a comprehensive outlook on D. radiodurans strategies of combating oxidative stress may open new avenues for antiaging and anticancer treatments. The study of the antioxidation protection in D. radiodurans is therefore of considerable potential interest for medicine and public health.     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.     

Preserving Genome Integrity: The DdrA Protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

Identification of PprM: a modulator of the PprI-dependent DNA damage response in Deinococcus radiodurans

Deinococcus radiodurans possesses a DNA damage response mechanism that acts via the PprI protein to induce RecA and PprA proteins, both of which are necessary in conferring extreme radioresistance. In an effort to further delineate the nature of the DNA damage response mechanism in D. radiodurans, we set out to identify novel components of the PprI-dependent signal transduction pathway in response to radiation stress. Here we demonstrate the discovery of a novel regulatory protein, PprM (a modulator of the PprI-dependent DNA damage response), which is a homolog of cold shock protein (Csp). Disruption of the pprM gene rendered D. radiodurans significantly sensitive to γ-rays. PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from D. geothermalis and Thermus thermophilus. Purified PprM is present as a homodimer under physiological conditions, as the case with Escherichia coli CspD. The pprA pprM double-disruptant strain exhibited higher sensitivity than the pprA or pprM single disruptant strains, suggesting that PprM regulates other hitherto unknown protein(s) important for radioresistance besides PprA. This study strongly suggests that PprM is involved in the radiation response mediated by PprI in D. radiodurans.     

Roles of PprA,IrrE, and RecA in the resistance of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> to germicidal and environmentally relevant UV radiation

To study the role of different DNA repair genes in the resistance of Deinococcus radiodurans to mono- and polychromatic UV radiation, wild-type strain and knockout mutants in RecA, PprA, and IrrE of D. radiodurans were irradiated with UV-C (254 nm), UV-(A + B) (280–400 nm) and UV-A (315–400 nm) radiation, and survival was monitored. The strain deficient in recA was highly sensitive to UV-C radiation compared to the wild-type, but showed no loss of resistance against irradiation with UV-(A + B) and UV-A, while pprA and irrE-deficient strains exhibited elevated sensitivity to UV-A and UV-(A + B) radiation. These results suggest that the repair of DNA double-strand breaks is essential after treatment with highly energetic UV-C radiation, whereas protection from oxidative stress may play a greater role in resistance to environmentally relevant UV radiation.     

Distinct Roles of Ape1 Protein,an Enzyme Involved in DNA Repair,in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing

High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.     

The Deinococcus radiodurans SMC protein is dispensable for cell viability yet plays a role in DNA folding

Deinococcus radiodurans contains a highly condensed nucleoid that remains to be unaltered following the exposure to high doses of γ-irradiation. Proteins belonging to the structural maintenance of chromosome protein (SMC) family are present in all organisms and were shown to be involved in chromosome condensation, pairing, and/or segregation. Here, we have inactivated the smc gene in the radioresistant bacterium D. radiodurans, and, unexpectedly, found that smc null mutants showed no discernible phenotype except an increased sensitivity to gyrase inhibitors suggesting a role of SMC in DNA folding. A defect in the SMC-like SbcC protein exacerbated the sensitivity to gyrase inhibitors of cells devoid of SMC. We also showed that the D. radiodurans SMC protein forms discrete foci at the periphery of the nucleoid suggesting that SMC could locally condense DNA. The phenotype of smc null mutant leads us to speculate that other, not yet identified, proteins drive the compact organization of the D. radiodurans nucleoid.     

Exogenous NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γ-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP), which can potentially increase the level of intracellular NAD⁺, on the level of γ-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NADP in different doses immediately after IR causes an increased level of γ-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that there could be a relationship between intracellular NAD⁺ content and DNA damage response in vivo.     

Enhanced intestinal tumor multiplicity and grade in vivo after HZE exposure: mouse models for space radiation risk estimates

Carcinogenesis induced by space radiation is considered a major risk factor in manned interplanetary and other extended missions. The models presently used to estimate the risk for cancer induction following deep space radiation exposure are based on data from A-bomb survivor cohorts and do not account for important biological differences existing between high-linear energy transfer (LET) and low-LET-induced DNA damage. High-energy and charge (HZE) radiation, the main component of galactic cosmic rays (GCR), causes highly complex DNA damage compared to low-LET radiation, which may lead to increased frequency of chromosomal rearrangements, and contribute to carcinogenic risk in astronauts. Gastrointestinal (GI) tumors are frequent in the United States, and colorectal cancer (CRC) is the third most common cancer accounting for 10% of all cancer deaths. On the basis of the aforementioned epidemiological observations and the frequency of spontaneous precancerous GI lesions in the general population, even a modest increase in incidence by space radiation exposure could have a significant effect on health risk estimates for future manned space flights. Ground-based research is necessary to reduce the uncertainties associated with projected cancer risk estimates and to gain insights into molecular mechanisms involved in space-induced carcinogenesis. We investigated in vivo differential effects of γ-rays and HZE ions on intestinal tumorigenesis using two different murine models, ApcMin/+ and Apc1638N/+. We showed that γ- and/or HZE exposure significantly enhances development and progression of intestinal tumors in a mutant-line-specific manner, and identified suitable models for in vivo studies of space radiation–induced intestinal tumorigenesis.     

p38γ regulates UV-induced checkpoint signaling and repair of UV-induced DNA damage

In eukaryotic cells, DNA damage triggers activation of checkpoint signaling pathways that coordinate cell cycle arrest and repair of damaged DNA. These DNA damage responses serve to maintain genome stability and prevent accumulation of genetic mutations and development of cancer. The p38 MAPK was previously implicated in cellular responses to several types of DNA damage. However, the role of each of the four p38 isoforms and the mechanism for their involvement in DNA damage responses remained poorly understood. In this study, we demonstrate that p38γ, but not the other p38 isoforms, contributes to the survival of UV-treated cells. Deletion of p38γ sensitizes cells to UV exposure, accompanied by prolonged S phase cell cycle arrest and increased rate of apoptosis. Further investigation reveal that p38γ is essential for the optimal activation of the checkpoint signaling caused by UV, and for the efficient repair of UV-induced DNA damage. These findings have established a novel role of p38γ in UV-induced DNA damage responses, and suggested that p38γ contributes to the ability of cells to cope with UV exposure by regulating the checkpoint signaling pathways and the repair of damaged DNA.     

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.     

A model for repair of radiation-induced DNA double-strand breaks in the extreme radiophile Deinococcus radiodurans

The bacterium Deinococcus (formerly Micrococcus) radiodurans and other members of the eubacterial family Deinococaceae are extremely resistant to ionizing radiation and many other agents that damage DNA. Stationary phase D. radiodurans exposed to 1.0-1.5 Mrad γ-irradiation sustains >120 DNA double-strand breaks (dsbs) per chromosome; these dsbs are mended over a period of hours with 100% survival and virtually no mutagenesis. This contrasts with nearly all other organisms in which just a few ionizing radiation induced-dsbs per chromosome are lethal. In this article we present an hypothesis that resistance of D. radiodurans to ionizing radiation and its ability to mend radiation-induced dsbs are due to a special form of redundancy wherein chromosomes exist in pairs, linked to each other by thousands of four-stranded (Holliday) junctions. Thus, a dsb is not a lethal event because the identical undamaged duplex is nearby, providing an accurate repair template. As addressed in this article, much of what is known about D. radiodurans suggests that it is particularly suited for this proposed novel form of DNA repair.     

Complex repair kinetics of DNA strand breaks induced by γ-rays or UV radiation in Ehrlich ascites tumour cells

Fluorometric analysis of DNA unwinding (FADU) – a sensitive technique for the detection of strand breaks in DNA – has been modified and used for the detailed investigation of repair kinetics of DNA-strand breaks arising under different conditions in Ehrlich ascites tumour (EAT) cells irradiated by γ-rays or ultraviolet (UV) radiation. The repair kinetics of DNA-strand breaks induced in EAT cells by γ-radiation was measured at radiation doses of 8, 20 and 50 Gy. We found complex repair curves in all cases, probably reflecting the combined processes of break rejoining and break generation during repair. In order to affect the above-mentioned processes, we have used different conditions of repair and different types of radiation. Lowering of the temperature of incubation and treating the cells by 5-fluoro-2′-deoxyuridine (FUdR) lead to complex changes of the repair curve with a reduced ``wave' pattern. In order to change the type of damage to DNA, we used UV radiation (254 nm, 10 and 20 J/m²). Detailed studies of the repair kinetics showed that the repair curve for 10 J/m² had a second maximum within 70 min after irradiation. Received: 17 May 1995 / Accepted in revised form: 15 March 1996     

Release of nascent trp mRNA from the operon DNA in chloramphenicol-treated cells of Escherichia coli.

Summary A synergistic effect of combined UV and -ray exposure was observed for inactivation of wild-type Schizosaccharomyces pombe. A recombinational repair process, known to be important in restitution of damage induced by both radiations, appears to be involved; a radiation-sensitive mutant defective in this repair pathway showed essentially no synergistic interaction between UV and -rays.Recovery from the synergistic effect of pre-exposure in wild-type cells did not display the expected fast -recovery and slow UV-recovery kinetics previously observed for regain of resistance to further exposure to the same radiation. Rather, UV-irradiated cells recovered quickly from synergistic inactivation on subsequent -exposure, while -irradiated cells recovered UV-resistance slowly. Recovery from synergism thus appears to reflect the nature of the second, and not the initial, radiation.AECL Reference No. 6154