Effects of radiation quality on interactions between oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation damages DNA and also induces oxidative stress, which can affect the function of proteins involved in DNA repair, thereby causing repair of DNA damage to become less efficient. We previously developed a mathematical model of this potentially synergistic relationship and applied it to γ-ray exposure data on the radiation-resistant prokaryote Deinococcus radiodurans. Here, we investigate the effects of radiation quality on these processes by applying the model to data on exposures of D. radiodurans to heavy ions with linear energy transfer (LET) of 18.5–11,300 keV/μm. The model adequately describes these data using three parameters combinations: radiogenic DNA damage induction, repair protein inactivation and cellular repair capacity. Although statistical uncertainties around best-fit parameter estimates are substantial, the behaviors of model parameters are consistent with current knowledge of LET effects: inactivation cross-sections for both DNA and proteins increase with increasing LET; DNA damage yield per unit of radiation dose also increases with LET; protein damage per unit dose tends to decrease with LET; DNA and especially protein damage yields are reduced when cells are irradiated in the dry state. These results suggest that synergism between oxidative stress and DNA damage may play an important role not only during γ-ray exposure, but during high-LET radiation exposure as well.     

Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.     

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.     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.     

Salt shield: intracellular salts provide cellular protection against ionizing radiation in the halophilic archaeon, Halobacterium salinarum NRC-1

The halophilic archaeon Halobacterium salinarum NRC-1 was used as a model system to investigate cellular damage induced by exposure to high doses of ionizing radiation (IR). Oxidative damages are the main lesions from IR and result from free radicals production via radiolysis of water. This is the first study to quantify DNA base modification in a prokaryote, revealing a direct relationship between yield of DNA lesions and IR dose. Most importantly, our data demonstrate the significance of DNA radiation damage other than strand breaks on cell survival. We also report the first in vivo evidence of reactive oxygen species scavenging by intracellular halides in H. salinarum NRC-1, resulting in increased protection against nucleotide modification and carbonylation of protein residues. Bromide ions, which are highly reactive with hydroxyl radicals, provided the greatest protection to cellular macromolecules. Modified DNA bases were repaired in 2 h post irradiation, indicating effective DNA repair systems. In addition, measurements of H. salinarum NRC-1 cell interior revealed a high Mn/Fe ratio similar to that of Deinococcus radiodurans and other radiation-resistant microorganisms, which has been shown to provide a measure of protection for proteins against oxidative damage. The work presented here supports previous studies showing that radiation resistance is the product of mechanisms for cellular protection and detoxification, as well as for the repair of oxidative damage to cellular macromolecules. The finding that not only Mn/Fe but also the presence of halides can decrease the oxidative damage to DNA and proteins emphasizes the significance of the intracellular milieu in determining microbial radiation resistance.     

Hypothetical Proteins Present During Recovery Phase of Radiation Resistant Bacterium Deinococcus radiodurans are Under Purifying Selection

Deinococcus radiodurans has an unusual capacity to recover from intense doses of ionizing radiation. The DNA repair proteins of this organism play an important role in repairing the heavily damaged DNA by employing a novel mechanism of DNA double-strand break repair. An earlier report stated that genes of many of these repair proteins are under positive selection implying that these genes have a tendency to mutate, which in turn provides selective advantage to this bacterium. Several “hypothetical proteins” are also present during the recovery phase and some of them have also been shown for their roles in radiation resistance. Therefore, we tested the selection pressure on the genes encoding these poorly characterized proteins. Our results show that a number of “hypothetical proteins” present during the repair phase have structural adaptations compared to their orthologs and the genes encoding them as well as those for the DNA repair proteins present during this phase are under purifying selection. Evidence of purifying selection in these hypothetical proteins suggests that certain novel characteristics among these proteins are conserved and seem to be under functional constraints to perform important functions during recovery process after gamma radiation damage.     

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K_d = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.     

A comparison of radiation-induced mitochondrial damage between neural progenitor stem cells and differentiated cells

Mitochondria play a key role in maintaining cellular homeostasis during stress responses, and mitochondrial dysfunction contributes to carcinogenesis, aging, and neurologic disease. We here investigated ionizing radiation (IR)-induced mitochondrial damage in human neural progenitor stem cells (NSCs), their differentiated counterparts and human normal fibroblasts. Long-term fractionated radiation (FR) with low doses of X-rays for 31 d enhanced mitochondrial activity as evident by elevated mitochondrial membrane potential (ΔΨm) and mitochondrial complex IV (cytochrome c oxidase) activity to fill the energy demands for the chronic DNA damage response in differentiated cells. Subsequent reduction of the antioxidant glutathione via continuous activation of mitochondrial oxidative phosphorylation caused oxidative stress and genomic instability in differentiated cells exposed to long-term FR. In contrast, long-term FR had no effect on the mitochondrial activity in NSCs. This cell type showed efficient DNA repair, no mitochondrial damage, and resistance to long-term FR. After high doses of acute single radiation (SR) (> 5 Gy), cell cycle arrest at the G2 phase was observed in NSCs and human fibroblasts. Under this condition, increase in mitochondria mass, mitochondrial DNA, and intracellular reactive oxygen species (ROS) levels were observed in the absence of enhanced mitochondrial activity. Consequently, cellular senescence was induced by high doses of SR in differentiated cells.

In conclusion, we demonstrated that mitochondrial radiation responses differ according to the extent of DNA damage, duration of radiation exposure, and cell differentiation.     

抗辐射菌Deinococcus radiodurans的多样性

抗辐射菌Deinococcus radiodurans是一种对电离辐射和其他DNA损伤因子具有极强抵抗能力的细菌,是研究DNA损伤与修复的模式生物。综述了国内外在抗辐射菌研究上取得的最新研究成果,从生存环境、对DNA损伤因子的抗性、抗性机理及其损伤修复关联基因等方面报道了抗辐射菌的多样性,并探讨了该细菌高效正确的DNA损伤修复机理的相关研究成果在生命科学、农业、环境修复及医学等领域的应用前景。     

Identification of SUMO modification sites in the base excision repair protein,Ntg1

DNA damaging agents are a constant threat to genomes in both the nucleus and the mitochondria. To combat this threat, a suite of DNA repair pathways cooperate to repair numerous types of DNA damage. If left unrepaired, these damages can result in the accumulation of mutations which can lead to deleterious consequences including cancer and neurodegenerative disorders. The base excision repair (BER) pathway is highly conserved from bacteria to humans and is primarily responsible for the removal and subsequent repair of toxic and mutagenic oxidative DNA lesions. Although the biochemical steps that occur in the BER pathway have been well defined, little is known about how the BER machinery is regulated. The budding yeast, Saccharomyces cerevisiae is a powerful model system to biochemically and genetically dissect BER. BER is initiated by DNA N-glycosylases, such as S. cerevisiae Ntg1. Previous work demonstrates that Ntg1 is post-translationally modified by SUMO in response to oxidative DNA damage suggesting that this modification could modulate the function of Ntg1. In this study, we mapped the specific sites of SUMO modification within Ntg1 and identified the enzymes responsible for sumoylating/desumoylating Ntg1. Using a non-sumoylatable version of Ntg1, ntg1ΔSUMO, we performed an initial assessment of the functional impact of Ntg1 SUMO modification in the cellular response to DNA damage. Finally, we demonstrate that, similar to Ntg1, the human homologue of Ntg1, NTHL1, can also be SUMO-modified in response to oxidative stress. Our results suggest that SUMO modification of BER proteins could be a conserved mechanism to coordinate cellular responses to DNA damage.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

End-damage-specific proteins facilitate recruitment or stability of X-ray cross-complementing protein 1 at the sites of DNA single-strand break repair

Ionizing radiation, oxidative stress and endogenous DNA-damage processing can result in a variety of single-strand breaks with modified 5' and/or 3' ends. These are thought to be one of the most persistent forms of DNA damage and may threaten cell survival. This study addresses the mechanism involved in recognition and processing of DNA strand breaks containing modified 3' ends. Using a DNA-protein cross-linking assay, we followed the proteins involved in the repair of oligonucleotide duplexes containing strand breaks with a phosphate or phosphoglycolate group at the 3' end. We found that, in human whole cell extracts, end-damage-specific proteins (apurinic/apyrimidinic endonuclease 1 and polynucleotide kinase in the case of 3' ends containing phosphoglycolate and phosphate, respectively) which recognize and process 3'-end-modified DNA strand breaks are required for efficient recruitment of X-ray cross-complementing protein 1-DNA ligase IIIalpha heterodimer to the sites of DNA repair.     

Oxidative DNA damage processing in nuclear and mitochondrial DNA

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, to examine the repair of different types of oxidative lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage.     

Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress.

Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative pentose phosphate cycle, regulates the NADPH/NADP(+) ratio in eukaryotic cells. G6PD deficiency is one of the most common mutations in humans and is known to cause health problems for hundreds of millions worldwide. Although it is known that decreased G6PD functionality can result in increased susceptibility to oxidative stress, the molecular targets of this stress are not known. Using a Chinese hamster ovary G6PD-null mutant, we previously demonstrated that exposure to a thiol-specific oxidant, hydroxyethyldisulfide, caused enhanced radiation sensitivity and an inability to repair DNA double strand breaks. We now demonstrate a molecular mechanism for these observations: the direct inhibition of DNA end binding activity of the Ku heterodimer, a DNA repair protein, by oxidation of its cysteine residues. Inhibition of Ku DNA end binding was found to be reversible by treatment of the nuclear extract with dithiothreitol, suggesting that the homeostatic regulation of reduced cysteine residues in Ku is a critical function of G6PD and the oxidative pentose cycle. In summary, we have discovered a new layer of DNA damage repair, that of the functional maintenance of repair proteins themselves. In view of the rapidly escalating number of roles ascribed to Ku, these results may have widespread ramifications.     

Cellular signal adaptation with damage control at low doses versus the predominance of DNA damage at high doses

Ionizing radiation is known to potentially interfere with cellular functions at all levels of cell organization and induces DNA lesions apparently with an incidence linearly related to D, also at low doses. On the other hand, low doses have also been observed to initiate a slowly appearing temporary protection against causation and accumulation of DNA lesions, involving the radical detoxification system, DNA repair and removal of DNA damage. This protection apparently does not operate at high doses; it has been described to be nonlinear, increasing initially with D, beginning to decrease when D exceeds approximately 0.1-0.2 Gy, and eventually disappearing at higher D. The various adaptive responses have been shown to last individually from hours to weeks in different cell types and resemble responses to oxidative stress. Damage to DNA is continuously and endogenously produced mainly by reactive oxygen species (ROS) generated in a normal oxidative metabolism. This endogenous DNA damage quantitatively exceeds DNA damage from low-dose irradiation, by several orders of magnitude. Thus, the protective responses following acute low-dose irradiation may be presumed to mainly counteract the endogenous DNA damage. Accordingly, the model described here uses two dose-effect functions, a linear one for causing and a nonlinear one for protecting against DNA damage from whatever cause in the irradiated cells and tissues. The resulting net dose-risk function strongly suggests that the incidence of cancer versus dose in the irradiated tissues is much less likely to be linear than to exhibit a threshold. The observed cancer incidence may even fall below the spontaneous incidence, when D to cells is below approximately 0.2 Gy. However incomplete, these data support a reexamination of the LNT hypothesis.     

The effect of aging on the DNA damage and repair capacity in 2BS cells undergoing oxidative stress

Aging is associated with a reduction in the DNA repair capacity under oxidative stress. However, whether the DNA damage and repair capacity can be a biomarker of aging remains controversial. In this study, we demonstrated two cause-and-effect relationships, the one is between the DNA damage and repair capacity and the cellular age, another is between DNA damage and repair capacity and the level of oxidative stress in human embryonic lung fibroblasts (2BS) exposed to different doses of hydrogen peroxide (H₂O₂). To clarify the mechanisms of the age-related reduction in DNA damage and repair capacity, we preliminarily evaluated the expressions of six kinds of pivotal enzymes involved in the two classical DNA repair pathways. The DNA repair capacity was observed in human fibroblasts cells using the comet assay; the age-related DNA repair enzymes were selected by RT-PCR and then verified by Western blot in vitro. Results showed that the DNA repair capacity was negatively and linearly correlated with (i) cumulative population doubling (PD) levels only in the group of low concentration of hydrogen peroxide treatment, (ii) with the level of oxidative stress only in the group of young PD cells. The mRNA expression of DNA polymerase δ1 decreased substantially in senescent cells and showed negative linear-correlation with PD levels; the protein expression level was well consistent with the mRNA level. Taken together, DNA damage and repair capacity can be a biomarker of aging. Reduced expression of DNA polymerase δ1 may be responsible for the decrease of DNA repair capacity in senescent cells.     

Elevated oxidative damage is correlated with reduced fitness in interpopulation hybrids of a marine copepod

Aerobic energy production occurs via the oxidative phosphorylation pathway (OXPHOS), which is critically dependent on interactions between the 13 mitochondrial DNA (mtDNA)-encoded and approximately 70 nuclear-encoded protein subunits. Disruptive mutations in any component of OXPHOS can result in impaired ATP production and exacerbated oxidative stress; in mammalian systems, such mutations are associated with ageing as well as numerous diseases. Recent studies have suggested that oxidative stress plays a role in fitness trade-offs in life-history evolution and functional ecology. Here, we show that outcrossing between populations with divergent mtDNA can exacerbate cellular oxidative stress in hybrid offspring. In the copepod Tigriopus californicus, we found that hybrids that showed evidence of fitness breakdown (low fecundity) also exhibited elevated levels of oxidative damage to DNA, whereas those with no clear breakdown did not show significantly elevated damage. The extent of oxidative stress in hybrids appears to be dependent on the degree of genetic divergence between their respective parental populations, but this pattern requires further testing using multiple crosses at different levels of divergence. Given previous evidence in T. californicus that hybridization disrupts nuclear/mitochondrial interactions and reduces hybrid fitness, our results suggest that such negative intergenomic epistasis may also increase the production of damaging cellular oxidants; consequently, mtDNA evolution may play a significant role in generating postzygotic isolating barriers among diverging populations.     

Slow accumulation of mutations in Xpc−/− mice upon induction of oxidative stress

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa^?/? and Xpc^?/? exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc^?/? mice, in contrary to Xpa^?/? and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc^?/? mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.