RESUMO
The ionizing radiation with high linear energy transfer (LET), such as a heavy ion beam, induces more serious biological effects than low LET ones, such as gamma- and X-rays. This indicates a difference in the DNA damage produced by low and high LET radiations and their biological effects. We have been studying the differences in DNA damage produced by gamma-rays and carbon ion beams. Therefore, we analyze mutations induced by both ionizing radiations to discuss the differences in their biological effects in this study. pUC19 plasmid DNA was irradiated by carbon ion beams in the solution containing 1M dimethyl sulfoxide to mimic a cellular condition. The irradiated DNA was cloned in competent cells of Escherichia coli. The clones harboring some mutations in the region of lacZα were selected, and the sequence alterations were analyzed. A one-deletion mutation is significant in the carbon-irradiated DNA, and the C:GâT:A transition is minor. On the other hand, the gamma-irradiated DNA shows mainly G:CâT:A transversion. These results suggest that carbon ion beams produce complex DNA damage, and gamma-rays are prone to single oxidative base damage, such as 8-oxoguanine. Carbon ion beams can also introduce oxidative base damage, and the damage species is 5-hydroxycytosine. This was consistent with our previous results of DNA damage caused by heavy ion beams. We confirmed the causal DNA damage by mass spectrometry for these mutations.
Assuntos
Carbono , Raios gama , Mutação , Carbono/química , Dano ao DNA , Íons Pesados , Transferência Linear de Energia , Escherichia coli/efeitos da radiação , Escherichia coli/genética , DNA/efeitos da radiaçãoRESUMO
There are various purification methods have been developed and applied to industrial wastewater with contaminated microorganisms. We previously reported that high-voltage pulsed discharge plasma with cavitation effectively kills Escherichia coli cells. We attempted to expand the application of this disinfection method by using microorganisms such as Bacillus subtilis, Deinococcus radiodurans, and Schizosaccharomyces pombe. These microbial cells were treated with the discharge plasma, and the cell viability, DNA damage, and morphological changes were analyzed to evaluate the bactericidal effect. Interestingly, D. radiodurans, a radio-resistant bacterium showed relatively high sensitivity to the discharge plasma. On the other hand, B. subtilis and S. pombe showed the resistance, showing both sporogenesis. The amount of DNA damage in the treated cells corresponded to the cell viability, but most of the treated cells did not show any morphological changes.
Assuntos
Desinfecção , Água , Bacillus subtilis , Desinfecção/métodos , Escherichia coli/genéticaRESUMO
Radon inhalation decreases the level of lipid peroxide (LPO); this is attributed to the activation of antioxidative functions. This activation contributes to the beneficial effects of radon therapy, but there are no studies on the risks of radon therapy, such as DNA damage. We evaluated the effect of radon inhalation on DNA damage caused by oxidative stress and explored the underlying mechanisms. Mice were exposed to radon inhalation at concentrations of 2 or 20 kBq/m3 (for one, three, or 10 days). The 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels decreased in the brains of mice that inhaled 20 kBq/m3 radon for three days and in the kidneys of mice that inhaled 2 or 20 kBq/m3 radon for one, three or 10 days. The 8-OHdG levels in the small intestine decreased by approximately 20-40% (2 kBq/m3 for three days or 20 kBq/m3 for one, three or 10 days), but there were no significant differences in the 8-OHdG levels between mice that inhaled a sham treatment and those that inhaled radon. There was no significant change in the levels of 8-oxoguanine DNA glycosylase, which plays an important role in DNA repair. However, the level of Mn-superoxide dismutase (SOD) increased by 15-60% and 15-45% in the small intestine and kidney, respectively, following radon inhalation. These results suggest that Mn-SOD probably plays an important role in the inhibition of oxidative DNA damage.
Assuntos
Dano ao DNA/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Radônio/farmacologia , Superóxido Dismutase/fisiologia , 8-Hidroxi-2'-Desoxiguanosina/análise , Administração por Inalação , Animais , Química Encefálica/efeitos da radiação , DNA Glicosilases/análise , Indução Enzimática/efeitos da radiação , Intestino Delgado/química , Intestino Delgado/efeitos da radiação , Rim/química , Rim/efeitos da radiação , Peroxidação de Lipídeos/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Oxirredução , Radônio/administração & dosagem , Radônio/uso terapêutico , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genéticaRESUMO
Radon inhalation activates antioxidative functions in mouse organs, thereby contributing to inhibition of oxidative stress-induced damage. However, the specific redox state of each organ after radon inhalation has not been reported. Therefore, in this study, we evaluated the redox state of various organs in mice following radon inhalation at concentrations of 2 or 20 kBq/m3 for 1, 3 or 10 days. Scatter plots were used to evaluate the relationship between antioxidative function and oxidative stress by principal component analysis (PCA) of data from control mice subjected to sham inhalation. The results of principal component (PC) 1 showed that the liver and kidney had high antioxidant capacity; the results of PC2 showed that the brain, pancreas and stomach had low antioxidant capacities and low lipid peroxide (LPO) content, whereas the lungs, heart, small intestine and large intestine had high LPO content but low antioxidant capacities. Furthermore, using the PCA of each obtained cluster, we observed altered correlation coefficients related to glutathione, hydrogen peroxide and LPO for all groups following radon inhalation. Correlation coefficients related to superoxide dismutase in organs with a low antioxidant capacity were also changed. These findings suggested that radon inhalation could alter the redox state in organs; however, its characteristics were dependent on the total antioxidant capacity of the organs as well as the radon concentration and inhalation time. The insights obtained from this study could be useful for developing therapeutic strategies targeting individual organs.
Assuntos
Especificidade de Órgãos/efeitos da radiação , Radônio/administração & dosagem , Administração por Inalação , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Oxirredução/efeitos da radiação , Análise de Componente Principal , Superóxido Dismutase/metabolismoRESUMO
DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA , DNA/metabolismo , Desoxirribonucleotídeos/genética , Proteínas/metabolismo , Recombinação Genética , Animais , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteína BRCA2/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromossomos/metabolismo , Cricetinae , DNA/química , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Decitabina , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Formaldeído/farmacologia , Histonas/metabolismo , Humanos , Peso Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/química , Rad51 Recombinase/metabolismoRESUMO
DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. We have recently shown that nucleotide excision repair (NER) and RecBCD-dependent homologous recombination (HR) collaboratively alleviate the lethal effect of DPCs in Escherichia coli. In this study, to gain further insight into the damage-processing mechanism for DPCs, we assessed the sensitivities of a panel of repair-deficient E. coli mutants to DPC-inducing agents, including formaldehyde (FA) and 5-azacytidine (azaC). We show here that the damage tolerance mechanism involving HR and subsequent replication restart (RR) provides the most effective means of cell survival against DPCs. Translesion synthesis does not serve as an alternative damage tolerance mechanism for DPCs in cell survival. Elimination of DPCs from the genome relies primarily on NER, which provides a second and moderately effective means of cell survival against DPCs. Interestingly, Cho rather than UvrC seems to be an effective nuclease for the NER of DPCs. Together with the genes responsible for HR, RR, and NER, the mutation of genes involved in several aspects of DNA repair and transactions, such as recQ, xth nfo, dksA, and topA, rendered cells slightly but significantly sensitive to FA but not azaC, possibly reflecting the complexity of DPCs or cryptic lesions induced by FA. UvrD may have an additional role outside NER, since the uvrD mutation conferred a slight azaC sensitivity on cells. Finally, DNA glycosylases mitigate azaC toxicity, independently of the repair of DPCs, presumably by removing 5-azacytosine or its degradation product from the chromosome.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , DNA Bacteriano/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Azacitidina/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Replicação do DNA , DNA Bacteriano/química , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Formaldeído/farmacologia , Mutação , Ligação Proteica , Recombinação GenéticaRESUMO
Ionizing radiation induces multiple damaged sites (clustered damage) together with isolated lesions in DNA. Clustered damage consists of closely spaced lesions within a few helical turns of DNA and is considered to be crucial for understanding the biological consequences of ionizing radiation. In the present study, two types of DNA, supercoiled plasmid DNA and linear lambda DNA, were irradiated with gamma-rays, carbon ion beams, and iron ion beams, and the spectra and yield of isolated DNA damage and bistranded clustered DNA damage were fully analyzed. Despite using different methods for damage analysis, the experiments with plasmid and lambda DNA gave largely consistent results. The spectra of both isolated and clustered damage were essentially independent of the quality of the ionizing radiation used for irradiation. The yields of clustered damage as well as of isolated damage decreased with the different radiation beams in the order gamma> C > Fe, thus exhibiting an inverse correlation with LET [gamma (0.2 keV/microm) < C (13 keV/microm) < Fe (200 keV/microm)]. Consistent with in vitro data, the yield of chromosomal DNA DSBs decreased with increasing LET in Chinese hamster cells irradiated with carbon ion beams with different LETs, suggesting that the decrease in the yield of clustered damage with increasing LET is not peculiar to in vitro irradiation of DNA, but is common for both in vitro and in vivo irradiation. These results suggest that the adverse biological effect of the ionizing radiation is not simply accounted for by the yield of clustered DNA damage, and that the complexity of the clustered damage needs to be considered to understand the biological consequences of ionizing radiation.
Assuntos
Radioisótopos de Carbono , Dano ao DNA , DNA/efeitos da radiação , Raios gama , Radioisótopos de Ferro , Animais , Cricetinae , DNA de Cadeia Simples/efeitos da radiação , Transferência Linear de Energia , Radiação IonizanteRESUMO
Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa-spermine cross-link adducts (Oxa-Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa-Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2-9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa-Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa-Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Óxido Nítrico/toxicidade , Nucleosídeos de Purina/metabolismo , Espermina/metabolismo , Sequência de Bases , Extratos Celulares , Adutos de DNA/metabolismo , Dano ao DNA , DNA Glicosilases/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismoRESUMO
vitamin D is 25-hydroxylated in the liver, before being activated by 1alpha-hydroxylation in the kidney. Recently, the rat cytochrome P450 2J3 (CYP2J3) has been identified as a principal vitamin D 25-hydroxylase in the rat [Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem 2004;279(22):22848-56]. In this study, we examine whether human CYP2J2 that exhibits 73% amino acid homology to rat CYP2J3 has similar catalytic properties. Recombinant human CYP2J2 was overexpressed in Escherichia coli, purified, and assayed for vitamin D 25-hydroxylation activity. We found significant 25-hydroxylation activity toward vitamin D3 (turnover number, 0.087 min(-1)), vitamin D2 (0.16 min(-1)), and 1alpha-hydroxyvitamin D3 (2.2 min(-1)). Interestingly, human CYP2J2 hydroxylated vitamin D2, an exogenous vitamin D, at a higher rate than it did vitamin D3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D3 (1.4 min(-1)) more efficiently than vitamin D2 (0.86 min(-1)). Our study demonstrated that human CYP2J2 exhibits 25-hydroxylation activity as well as rat CYP2J3, although the activity of human CYP2J2 is weaker than rat CYP2J3. CYP2J2 and CYP2J3 exhibit distinct preferences toward vitamin D3 and D2.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Animais , Sequência de Bases , Colestanotriol 26-Mono-Oxigenase/química , Sistema Enzimático do Citocromo P-450/química , Primers do DNA , Humanos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.
Assuntos
Dano ao DNA , DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA , Pentoxil (Uracila)/análogos & derivados , Uracila/análogos & derivados , Sequência de Aminoácidos , Catálise , DNA Glicosilases/genética , Análise Mutacional de DNA , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pentoxil (Uracila)/metabolismo , Homologia Estrutural de Proteína , Uracila/metabolismo , Uracila-DNA Glicosidase , Proteínas de XenopusRESUMO
Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (V(max)/K(m)) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [(3)H]Xan from the substrate. The treatment of E.coli with N-methyl-N'-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA(+) but not alkA(-) strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan.
Assuntos
DNA Glicosilases , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Guanina/metabolismo , N-Glicosil Hidrolases/metabolismo , Óxidos de Nitrogênio/toxicidade , Desoxirribonuclease (Dímero de Pirimidina) , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Guanina/química , Óxido Nítrico/química , Óxido Nítrico/toxicidade , Ácido Nitroso/química , Ácido Nitroso/toxicidade , Oligonucleotídeos/química , Nucleosídeos de Purina/química , Nucleosídeos de Purina/metabolismo , Xantina/química , Xantina/metabolismoRESUMO
Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm(-1), respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process.
Assuntos
Sobrevivência Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA/genética , DNA/efeitos da radiação , Íons Pesados , Animais , Sequência de Bases , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , DNA/química , Relação Dose-Resposta à Radiação , Transferência Linear de Energia , Dados de Sequência Molecular , Doses de RadiaçãoRESUMO
Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 microg/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.
Assuntos
Biotina/análogos & derivados , Dano ao DNA , Análise Mutacional de DNA/métodos , DNA-Formamidopirimidina Glicosilase/química , DNA/química , DNA/efeitos da radiação , Desoxirribonuclease (Dímero de Pirimidina)/química , Proteínas de Escherichia coli/química , Pareamento Incorreto de Bases/efeitos da radiação , DNA/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Relação Dose-Resposta à Radiação , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Doses de RadiaçãoRESUMO
Clustered DNA damage (locally multiply damaged site) is thought to be a critical lesion caused by ionizing radiation, and high LET radiation such as heavy ion particles is believed to produce high yields of such damage. Since heavy ion particles are major components of ionizing radiation in a space environment, it is important to clarify the chemical nature and biological consequences of clustered DNA damage and its relationship to the health effects of exposure to high LET particles in humans. The concept of clustered DNA damage emerged around 1980, but only recently has become the subject of experimental studies. In this article, we review methods used to detect clustered DNA damage, and the current status of our understanding of the chemical nature and repair of clustered DNA damage.
Assuntos
Radiação Cósmica , Dano ao DNA/fisiologia , Dano ao DNA/efeitos da radiação , Reparo do DNA/fisiologia , Reparo do DNA/efeitos da radiação , Íons Pesados , DNA Glicosilases , Eletroforese em Gel de Campo Pulsado , Endonucleases/efeitos da radiação , Humanos , Radical Hidroxila , Transferência Linear de Energia , Doses de Radiação , Radiação IonizanteRESUMO
Rubrobacter radiotolerans is the most radio-resistant eubacterium without spore-formation in the life cycle, and its D(37) is 16,000 Gy against gamma-rays. To understand the molecular mechanism of the high radio-resistance, we purified and characterized superoxide dismutase (SOD) of this organism as enzymatic radical scavenger, and then analyzed its genetic information. The purified SOD protein formed homo-tetramerization of 24,000 Da-monomer, while maintaining its enzymatic activity against potassium cyanide and hydrogen peroxide. We obtained a partial amino acid sequence of the protein and cloned the gene from it. Sequence analysis of the cloned gene indicated that the protein showed a similarity to other bacterial manganese SODs (Mn-SODs). Sequencing for adjacent regions of the gene showed that the gene had promoter elements with an open reading frame for putative PAS/PAC sensor protein at the 5'-adjacent region. Introduction of the gene into Escherichia coli cells lacking intrinsic SOD genes restored the cellular enzymatic activity and resistance to methyl viologen, indicating the gene at work. A mutant cell harboring this gene also became resistant against gamma-rays. The present results suggest that the protein in question is the Mn-SOD of R. radiotolerans, a good candidate as a radio-protection factor for this bacterial radio-resistance.
Assuntos
Actinobacteria/enzimologia , Actinobacteria/efeitos da radiação , Superóxido Dismutase/metabolismo , Actinobacteria/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Raios gama , Genes Bacterianos , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Tolerância a Radiação/genética , Tolerância a Radiação/fisiologia , Superóxido Dismutase/química , Superóxido Dismutase/genéticaRESUMO
Endogenous and environmental genotoxic agents produce DNA damage and induce cell death and mutations. The repair mechanisms of base lesions and single and double strand breaks have been well characterized in both prokaryotic and eukaryotic cells. However, the molecular pathways that repair or tolerate DNA-protein crosslinks (DPCs) remains to be largely elucidated. In this study, we constructed DNA substrates containing defined DPCs and assessed the incision activities of prokaryotic and eukaryotic nucleotide excision repair systems for DPCs in vitro.
Assuntos
Dano ao DNA , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , DNA/química , Células HeLa , Humanos , Proteínas/químicaRESUMO
DNA-protein crosslinks (DPCs) are extremely bulky DNA lesions, and steric hindrance imposed by covalently trapped proteins would hamper the transaction of DNA such as replication, transcription, and repair. However, it has been largely elusive how cells mitigate the genotoxic effect of DPCs. We have recently shown that nucleotide excision repair (NER) and homologous recombination (HR) differentially contribute to the repair of DPCs in E. coli cells. Several lines of genetic and biochemical evidence indicate that NER repairs DPCs with crosslinked proteins (CLPs) of sizes less than 12-14 kDa, whereas DPCs with oversized CLPs are processed exclusively by RecBCD-dependent HR. The present result shows that cells use the coordinated actions of NER and HR to deal with unusually bulky DNA lesions like DPCs.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Recombinação Genética , Reparo do DNA/genética , Escherichia coli/genética , Nucleosídeos de Purina/químicaRESUMO
Among numerous DNA damaging factors, ionizing radiation produces the damage showing very unique structure. Since ionizing radiation passes through a target DNA as a beam, the respective induced lesions locate close together around the track. Such damage aggregation on target DNA called "clustered DNA damage" is thought to be a major cause of the specific and serious effect of ionizing radiation. However, we have less knowledge about the structure of clustered DNA damage, which seems very important in its biological impact. Therefore, we evaluated procedure to analyze the structure of clustered DNA damage induced by ionizing radiation. In the present study, we used polyacrylamide gel electrophoresis to separate oligodeoxyribonucleotide (ODN) substrates containing clustered DNA damage with post-modification with aldehyde reactive probe. The designed procedure well counted the number of abasic site in the model substrate of clustered DNA damage.
Assuntos
Dano ao DNA , Oligodesoxirribonucleotídeos/efeitos da radiação , Radiação Ionizante , Carbono/química , Eletroforese em Gel de Poliacrilamida , Íons Pesados , Transferência Linear de Energia , Oligodesoxirribonucleotídeos/químicaRESUMO
DNA-protein cross-link (DPC) damage is produced by varieties of genotoxic agents such as ionizing radiation, UV light, aldehydes, and some chemotherapeutic compounds. DPCs would be harmful to cells since proteins immobilized on DNA strands could hamper DNA transactions in cells. However, it has not been clarified how DPCs are repaired in cells. In the present study, we prepared DNA substrates containing defined DPCs and tested them for repair enzymes. We also examined the sensitivity of Escherichia coli to DPC-inducing agents.