Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1093/jrr/rraa129

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Publishing type：Research paper (scientific journal)   Publisher：The Japanese Skin Cancer Society  

DOI： 10.5227/skincancer.34.50

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DOI： 10.1063/1.5092296

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOP PUBLISHING LTD  

Plasma-based techniques are expected to have practical use for wastewater purification with a potential for killing contaminated microorganisms and degrading recalcitrant materials. In the present study, we analysed oxidative DNA damage in bacterial cells treated by the plasma to unveil its mechanisms in the bactericidal process. Escherichia coli cell suspension was exposed to the plasma induced by applying an alternating-current voltage of about 1 kV with bubbling formed by water-cavitation, termed pulsed discharge with cavitation. Chromosomal DNA damage, such as double strand break (DSB) and oxidative base lesions, increased proportionally with the applied energy, as determined by electrophoretic and mass spectrometric analyses. Among the base lesions identified, the yields of 8-hydroxyguanine (8-OH-G) and 5-hydroxycytosine (5-OH-C) in chromosomal DNA increased by up to 4- and 15-fold, respectively, compared to untreated samples. The progeny DNA sequences, derived from plasmid DNA exposed to the plasma, indicated that the production rate of 5-OH-C exceeded that of 8-OH-G, as G:C to A:T transitions accounted for 65% of all base changes, but only a few G:C to T:A transversions were observed. The cell viabilities of E. coli cells decreased in direct proportion to increases in the applied energy. Therefore, the plasma-induced bactericidal mechanism appears to relate to oxidative damage caused to bacterial DNA. These results were confirmed by observing the generation of hydroxyl radicals and hydrogen peroxide molecules following the plasma exposure. We also compared our results with the plasma to those obtained with Cs-137 gamma-rays, as a well-known ROS generator to confirm the DNA-damaging mechanism involved.

DOI： 10.1088/0022-3727/48/36/365401

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Institute of Electrical Engineers of Japan  

In this study, we propose a new treatment method to sanitize ballast water contaminated with plankton and/or bacteria. This method is constituted of water cavitation and discharge plasma in this cavitation field. The 0.5% brackish water containing brine shrimp (2.5 × 10&lt
sup&gt
4&lt
/sup&gt
and 1.25 × 10&lt
sup&gt
5&lt
/sup&gt
m&lt
sup&gt
-3&lt
/sup&gt
) was treated in 60 s. Then, we saw no surviving plankton in the treated water. Similarly, the treatment of water containing Escherichia coli cells (4 × 10&lt
sup&gt
15&lt
/sup&gt
m&lt
sup&gt
-3&lt
/sup&gt
) in 120 s completed the sterilization. We also observed the DNA damage and destruction of the cellular morphology after treatment. It seems that the mechanisms of sterilization are radical reactions and physical damage caused by shockwave, and radical reactions are dominant mechanism.

DOI： 10.1541/ieejfms.135.357

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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 mu m(-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.

DOI： 10.1093/jrr/rru122

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Pulsed discharge is used for sterilization and disinfection, but the details of the molecular mechanisms remain largely unknown. Since pulsed discharge generates reactive oxygen species (ROS), we analyzed the oxidative DNA damages after pulsed discharge treatment to consider the involvement of ROS in the damaging process. We applied pulsed discharge with cavitation to plasmid DNA molecules and estimated the yields of the damages by agarose gel electrophoresis. The treated DNA contained various oxidative DNA damages, including single and double strand breaks and base lesions. The yields of the damages increased in response to the energy used for pulsed discharge. We also measured the yield of 8hydroxyguanine (8-OH-G), one of the major oxidative base lesions, in the treated plasmid DNA by mass spectrometry quantitatively and found that the yield of the oxidative base lesion corresponded to the increment of the applied energy. In addition, we observed the involvement of mutM gene, which is responsible for repair of 8-OH-G, in the increased sensitivity of Estherichia coil to pulsed discharge. Therefore, ROS seem to mediate the sterilization ability of pulsed discharge. (C) 2014 Published by Elsevier B.V.

DOI： 10.1016/j.elstat.2014.10.010

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DOI： 10.1541/ieejfms.135.357

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Authorship：Corresponding author   Language：Japanese   Publisher：Japanese Society of Radiation Safety Management  

The Saga Medical School Radiation Facility was founded in 1980, and is now The Division of Radioactive Compound Utility of Analytical Research Center for Experimental Sciences, Saga University. The facility provides laboratories to handle unsealed radioactive materials and gamma- and X-rays irradiating apparatuses for our faculties and students. We reduced the scale of this facility in 2012, because the use of the facility has decreased during the last two decades. The number of users in the last year was only a fifth of the peak one in 1995. The renovated facility has two-thirds of its floor space and some remodelings including removes of the inside lavatories and the disposal for radioactive waste organic solution. We here report detail of this renovation and the related approvals.

DOI： 10.11269/jjrsm.13.62

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Promotion of bulb germination using pulsed power has been demonstrated in our previous experiments. In order to clear the mechanisms of promotion of germination, the glucose concentration of bulb was measured at with and without pulsed power. The value of difference at between with and without pulsed power was about 2 μg/g. From the analysis of glucose, it was found that the pulsed power contributes to the high concentration of glucose, and it promotes germination of bulb. © 2013 The Institute of Electrical Engineers of Japan.

DOI： 10.1541/ieejfms.133.64

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DOI： 10.1541/ieejfms.133.64

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN RADIATION RESEARCH SOC  

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 homotetramerization 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.

DOI： 10.1269/jrr.11105

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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.

DOI： 10.1074/jbc.M109.019174

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

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.

DOI： 10.1128/JB.00417-09

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN RADIATION RESEARCH SOC  

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/mu m) < C (13 keV/mu m) < Fe (200 keV/mu m)]. 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.

DOI： 10.1269/jrr.07089

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Language：Japanese   Publisher：放射線生物研究会  

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Other Link： http://search.jamas.or.jp/link/ui/2008168543

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1269/jrr.07089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strandblock the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCDdependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.

DOI： 10.1016/j.molcel.2007.07.029

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We investigated whether or not social stress (isolation, 1 mouse per cage) increases oxidative DNA damage in mouse peripheral blood cells. Male BALB/c mice (4 weeks old) were housed 5 per cage for 10 days. After acclimatization, mice were exposed to isolation stress for 7 and 30 days. Control mice were housed 5 per cage. Serum levels of corticosterone, which is a well known stress marker, and antioxidant compounds, ascorbic acid and a-tocopherol, were determined by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and ESACoul Array analysis, respectively. Single cell gel electrophoresis (comet assay) using formamidopyrimidine DNA glycosylase (FPG) was done to determine oxidative DNA damage in mouse peripheral blood cells. The significant increases of plasma level of corticosterone were observed in mice exposed to isolation stress for 7 days and 30 days. Although no significant differences in plasma concentration of ascorbic acid and a-tocopherol were observed between the control mice and the isolated mice, oxidative DNA damage was induced in the isolated mice for 7 days and 30 days. These results suggest that social stress, isolation, causes mild oxidation in mice. Key words: social stress, isolation, oxidative DNA damage, comet assay. © 2007, The Japanese Environmental Mutagen Society. All rights reserved.

DOI： 10.3123/jemsge.29.17

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Vitamin D is 25-hydroxylated in the liver, before being activated by 1 alpha-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 D-3 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 D-3 (turnover number, 0.087 min(-1)), vitamin D-2 (0.16 min(-1)), and 1 alpha-hydroxyvitamin D-3 (2.2 min(-1)). Interestingly, human CYP2J2 hydroxylated vitamin D-2, an exogenous vitamin D, at a higher rate than it did vitamin D-3, an endogenous vitamin D, whereas, rat CYP2J3 hydroxylated vitamin D-3 (1.4 min(-1)) more efficiently than vitamin D-2 (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 D-3 and D-2. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.steroids.2006.04.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

It has been suggested that carcinogenesis associated with chronic inflammation involves DNA damage by nitric oxide ( NO) and other reactive species secreted from macrophages and neutrophils. The guanine moiety of DNA reacts with NO, yielding two major deamination products: xanthine (Xan) and oxanine (Oxa). Oxa reacts further with polyamines and DNA binding proteins to form cross-link adducts. In the present study, we characterized the structure of the cross-link adducts of Oxa with spermine (Oxa-Sp). Spectrometric analysis of Oxa-Sp adducts showed that they are ring-opened adducts of Oxa covalently bonded to the terminal amino ( major product) and internal imino ( minor product) groups of spermine. To assess genotoxic potential, Xan, Oxa, Oxa-Sp and an abasic (AP) site were site specifically incorporated into oligonucleotide templates. These lesions differentially blocked in vitro DNA synthesis catalyzed by DNA polymerase I Klenow fragment (Pol I Kf). The relative efficiency of translesion synthesis was G (1) &GT; Oxa (0.19) &GT; Xan (0.12) &GT; AP (0.088) &GT; Oxa-Sp (0.035). Primer extension assays with a single nucleotide and Pol I Kf revealed that non-mutagenic dCMP was inserted most efficiently opposite Xan and Oxa, with the extent of primer elongation being 65% for Xan and 68% for Oxa. However, mutagenic nucleotides were also inserted. The extent of primer elongation for Xan was 16% with dTMP and 14% with dGMP, whereas that for Oxa was 49% with dTMP. For Oxa-Sp, mutagenic dAMP (13%) was preferentially inserted. Accordingly, when generated in vivo, Xan and Oxa would constitute moderate blocks to DNA synthesis and primarily elicit G: C to A: T transitions when bypassed, whereas Oxa-Sp would strongly block DNA synthesis and elicit G: C to T: A transversions.

DOI： 10.1093/mutage/gei027

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publisher：The Society of Sea Water Science, Japan  

Abolition of the salt monopoly has lead to increases in the distribution and the import of natural salts and foods containing these salts. There is a possibility of the contaminations of these natural salt products with halophiles such as extremely halophilic archaea under the circumstances. The contaminants can putrefy the salt products, and damage the food manufacturers and importers dealing in salts. Therefore, we were convinced of the necessity of detection procedures for contaminated halophiles, and examined the conditions for detecting halophiles specifically using a polymerase chain reaction (PCR) in the present study. Three pairs of oligodeoxyribonucleotides (ODN) reported previously and novel twelve pairs of ODN were constructed for PCR primers. The latter were derived from twelve insertion sequences of halophile (ISH) in the whole genome of Halobacterium sp. NRC-1. The chromosomal DNA of two extremely halophilic archaea, Halobacterium sp. NRC-1and H. salinarum, and four other food contaminating eubacteria such as Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Vibrio parahaemolyticus were extracted and purified for PCR templates. The amplified products were analyzed on agarose gel electrophoreses.The results indicated that the four pairs of PCR primers showed halophile-specific amplification. It is suggested that these four pairs of primers are useful for the specific detection of the contaminating halophiles in natural salt products.

DOI： 10.11457/swsj1965.59.61

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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.

DOI： 10.1093/nar/gki513

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：MEDIMOND S R L  

Recombinant rat CYP2J3 and human CYP2J2 were successfully expressed in the E. coli expression system by co-expression of chaperone proteins. Purified proteins were applied to analysis of catalytic activities toward vitamin D compounds using a reconstituting system containing NADPH-P450 reductase. The CYP2J2 showed significant but about 10 times lower 25-hydroxylation activities than CYP2J3 toward vitamin D-3 and 1 alpha-OH-D-3. Interestingly, CYP2J2 prefers vitamin D-2 to vitamin D-3 as a substrate, whereas CYP2J3 did vitamin D-3 to vitamin D-2, The results suggest involvement of CYP2J subfamily proteins in vitamin D activation through 25-hydroxylation reaction, although they showed distinct preferences and activities toward vitamin D compounds.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

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.

DOI： 10.1093/nar/gkh859

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN RADIATION RESEARCH SOC  

Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 mug/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.

DOI： 10.1269/jrr.45.229

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In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2). In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates. hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg:5S-Tg was 13:1 for hNTH1 and 1.5:1 for hNEIL1. This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg:5S-Tg was 1:2.5 for Endo III and 3.2:1 for Endo VIII. Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates. In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal. hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the -fold of the active site of the Endo VIII homologues. The profiles of the excision of the Tg isomers with HeLa and E. coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells. However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells.

DOI： 10.1074/jbc.M400393200

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.2187/bss.18.206

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Chronic inflammation is a risk factor for many human cancers, and nitric oxide ( NO) produced in inflamed tissues has been proposed to cause DNA damage via nitrosation or oxidation of base moieties. Thus, NO-induced DNA damage could be relevant to carcinogenesis associated with chronic inflammation. In this report, we report a novel genotoxic mechanism of NO that involves DNA-protein cross-links (DPCs) induced by oxanine (Oxa), a major NO-induced guanine lesion. When a duplex DNA containing Oxa at the site-specific position was incubated with DNA-binding proteins such as histone, high mobility group (HMG) protein, and DNA glycosylases, DPCs were formed between Oxa and protein. The rate of DPC formation with DNA glycosylases was approximately two orders of magnitude higher than that with histone and HMG protein. Analysis of the reactivity of individual amino acids to Oxa suggested that DPC formation occurred between Oxa and side chains of lysine or arginine in the protein. A HeLa cell extract also gave rise to two major DPCs when incubated with DNA-containing Oxa. These results reveal a dual aspect of Oxa as causal damage of DPC formation and as a suicide substrate of DNA repair enzymes, both of which could pose a threat to the genetic and structural integrity of DNA, hence potentially leading to carcinogenesis.

DOI： 10.1074/jbc.M212847200

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5-Formyluracil (fU) is a major oxidative thymine lesion produced by reactive oxygen species and exhibits genotoxic and cytotoxic effects via several mechanisms. In the present study, we have searched for and characterized mammalian fU-DNA glycosylase (FDG) using two approaches. In the first approach, the FDG activity was examined using purified base excision repair enzymes. Human and mouse endonuclease III homologues (NTH1) showed a very weak FDG activity, but the parameter analysis and NaBH4 trapping assays of the Schiff base intermediate revealed that NTH1 was kinetically incompetent for repair of W. In the second approach, FDG was partially purified (160-fold) from rat liver. The enzyme was a monofunctional DNA glycosylase and recognized fU in single-stranded (ss) and double-stranded (ds) DNA. The most purified FDG fraction also exhibited monofunctional DNA glycosylase activities for uracil (U), 5-hydroxyuracil (hoU), and 5-hydroxymethyluracil (hmU) in ssDNA and dsDNA. The fU-excising activity of FDG was competitively inhibited by dsDNA containing U-G, hoU(.)G, and hmU(.)A but not by intact dsDNA containing T-A. Furthermore, the activities of FDG for fU, hmU, hoU, and U in ssDNA and dsDNA were neutralized by the antibody raised against SMUG1 uracil-DNA glycosylase, showing that FDG is a rat homologue of SMUG1.

DOI： 10.1021/bi027322v

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In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.

DOI： 10.1021/bi0273213

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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 [H-3]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.

DOI： 10.1093/nar/gkf630

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2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3'-5' exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f(ins) = V(max)/K(m) for insertion) and extension (f(ext) = V(max)/K(m) for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f(ins) = 1) dTMP (2.4 x 10(-4)) approximately dAMP (8.1 x 10(-5)) > dGMP (4.5 x 10(-7))), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f(ext) = 1) > mFapyG:T (4.6 x 10(-3)) mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.

DOI： 10.1074/jbc.M200316200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In order to investigate the mechanism of the production of oxidative DNA damage by hyperglycemia, we measured formamidopyrimidine N-glycosylase (FPG)-sensitive sites by the comet assay in human umbilical vein endothelial cells (HUVECs) cultured under various conditions including high glucose.
Mean values of FPG-sensitive sites were higher in HUVECs cultured for 5 days in high glucose (45 mM) compared with normal glucose (5 mM) medium (P &lt; 0.001). FPG-sensitive sites increased in a time-dependent manner under high glucose treatment (3 days: P &lt; 0.05, 5 days: P &lt; 0.001), whereas L-glucose, which is taken up poorly into the cells, gave a slight increase in FPG-sensitive sites (P &lt; 0.05). Flow cytometric analysis using 6-carboxy-2 ' ,7 ' -dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) showed that incubation with L-glucose produced more reactive oxygen species than incubation with D-glucose. However, these increases were slight (1.22- and 1.12-folds, respectively).
Incubation of HUVECs with aminoguanidine (100 muM) or pyridoxamine (1 mM), which are inhibitors of glycation, decreased the levels of FPG-sensitive sites (P &lt; 0.001). However, these inhibitors did not suppress the intracellular generation of reactive oxygen species induced by high glucose. These results indicate that FPG-sensitive sites induced by high glucose are not due to intracellular reactive oxygen species.
In order to clarify what caused the induction of FPG-sensitive sites, we investigated the effect of glyoxal and 3-deoxyglucosone (3-DG) on the induction of FPG-sensitive sites and the intracellular production of reactive oxygen species in HUVECs. Glyoxal and 3-DG at a concentration of 100 mug/m induced FPG-sensitive sites (P &lt; 0.001, P &lt; 0.1, respectively). In contrast, glyoxal did not generate reactive oxygen species inside HUVECs. The results shown in this study suggest that glyoxal formed intracellularly or extracellularly during high glucose treatment might induce FPG-sensitive sites by a mechanism not involving reactive oxygen species. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0027-5107(01)00196-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3'-5'-exonuclease, Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f(A) = V-max/K-m) decreased to 1/4 to 1/2, depending on the nearest neighbor base pair, and that of dGMP (f(G)) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (f(G)/f(A) for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pK(a) = 8.6) but not T (pK(a) = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G, The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.

DOI： 10.1074/jbc.M008598200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

7,8-Dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells. Both lesions are repaired by human 7,8 dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively. In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The k(cat)/K-m values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg, However, the k(cat)/K-m values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg, Analysis of the Schiff base intermediate by NaBH4 trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases. Surprisingly, such preferences were almost abolished and less than a-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair. The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg. The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K-m), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.

DOI： 10.1074/jbc.275.7.4956

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN RADIATION RESEARCH SOC  

Rubrobacter radiotolerans is an extremely radioresistant bacterium. It exhibits higher resistance than the well-known radioresistant bacterium Deinococcus radiodurans, but the molecular mechanisms responsible for the radioresistance of R. radiotolerans remain unknown. In the present study, we have demonstrated the presence of a novel DNA repair enzyme in R. radiotolerans cells that recognizes radiation-induced DNA damages such as thymine glycol, urea residues, and abasic sites. The enzyme was purified from the crude cell extract by a series of chromatography to an apparent physical homogeneity. The purified enzyme showed a single band with a molecular mass of approximately 40 kDa in SDS-polyacrylamide gel electrophoresis, and was designated as R-endonuclease. R-Endonuclease exhibited repair activity for thymine glycol, urea residues, and abasic sites present in plasmid DNA, but did not act on intact DNA, UV-irradiated DNA and DNA containing reduced abasic sites. The substrate specificity together with the salt and pH optima suggests that R-endonuclease is a functional homolog of endonuclease III of Escherichia coli.

DOI： 10.1269/jrr.41.19

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Escherichia coli endonuclease III (Endo III) and its eukaryotic homologues are major repair enzymes for pyrimidine lesions formed by reactive oxygen species and ionizing radiation. In the present study, the activities of Endo III and its mouse homologue (mNTH1) have been compared using defined oligonucleotide substrates containing a urea residue (UR), two cis-thymine glycol (TG) diastereoisomers, 5,6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU). The substrates were incubated with Endo III and mNTH1, and their activities were compared based on the product analysis by gel electrophoresis. Endo III recognized all base lesions tested, but the activity for DHT was extremely lower than other substrates. In contrast, albeit some preference of UR, mNTH1 showed essentially comparable activities for all substrates including DHT. Comparison of the enzymatic parameters for cis-TG and DHT revealed that large decreases in the affinity (K-m, 27-fold) and k(cat) (11-fold) relative to cis-TG made DHT an very poor substrate for Endo III. mNTH1 had comparable affinities and k(cat) for both cis-TG and DHT, though turnover (k(cat)) of mNTH1 was notably slower than Endo III. In view of the reaction mechanism, the paired base effect on the damage recognition by the two enzymes was also examined. The activities of Endo III for UR and HOU were paired base-independent, but those for cis TG and DHT were significantly enhanced when paired with G. With mNTH1, the paired base effect was evident only for DHT. The variations of the repair activity with paired bases and enzymes are discussed in relation to the base flipping mechanism suggested for base excision repair enzymes.

DOI： 10.1021/bi000422l

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The activity of prokaryotic and mammalian thymine glycol (Tg) glycosylases including Escherichia coli endonuclease III (Endo III) and endonuclease VIII (Endo VIII) and mouse Endo III homologue (mNth1) for formamidopyrimidine (Fapy) has been investigated using defined oligonucleotide substrates, 2,6-Diamino-4-hydroxy-5-N-methylformamidopyrimidine, a methylated Fapy derived from guanine, was site specifically incorporated in the oligonucleotide, The substrates containing Fapy:N pairs (N = A, G, C, T) as well as a Tg:A pair, a physiological substrate of Endo HI, Endo VIII, and mNth1, were treated by the enzymes and nicked products were quantified by gel electrophoresis. The activity of Endo III and Endo VIII for Fapy varied markedly depending on the paired base, being the highest with G (activity relative to Tg = 0.55 (Endo III) and 0.41 (Endo VIII)) and the lowest with C (0.05 (Endo III) and 0.06 (Endo VIII)). In contrast, mNth1 recognized all Fapy pairs equally well and the activity was comparable to Tg, The results obtained in the nicking assay were further substantiated by the analysis of the Schiff base intermediate using NaBH4 trapping assays. These results indicate that Escherichia coli and mammalian Tg glycosylases have a potential activity to recognize Fapy. However, as demonstrated for Fapy:C pairs, their distinctive activities implicate unequal participation in the repair of Fapy lesions in cells.

DOI： 10.1074/jbc.M000576200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. H-1 and C-13 NMR chemical shifts of 5-formyl-2'-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Robayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J. Biol. Chem. 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

DOI： 10.1074/jbc.274.35.25136

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication. In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H. (1999) J. Biol. Chem. 274, 25136-25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates, Mispairing frequency of fU was determined by incorporation of 2'-deoxyribonucleoside 5'-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3'-5' exonuclease. Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2-12 times higher than that of thymine at pH 7.8 and 2.6-6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer), AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable. In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G; mispairs, but it did not recognize fU:A pairs. Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU, These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS, Biological relevance of the present results is discussed in light of DNA replication and repair in cells.

DOI： 10.1074/jbc.274.35.25144

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GUSTAV FISCHER VERLAG  

Halobacterium salinarium exhibits resistance to ultraviolet (UV) and ionizing radiation. This organism contains carotenoids and also accumulates highly concentrated KCl in the cell. In the present study, DNA lesions generated by UV and ionizing radiation were measured in vitro in the presence and absence of bacterioruberin, the major carotenoid of H. salinarium, and KCl to elucidate their influences on DNA damage production. When plasmid DNA (pDEL19) was UV-irradiated, formation of cyclobutane pyrimidine dimers (CPD) was slightly suppressed by 0.1 mM bacterioruberin. The same concentration of bacterioruberin suppressed the formation of DNA single strand breaks (SSB) by ionizing radiation more efficiently. The formation of CPD by UV and SSB by ionizing radiation was also repressed by 2M KCl but protection against ionizing radiation was extremely efficient.

DOI： 10.1016/S0944-5013(99)80013-5

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：GUSTAV FISCHER VERLAG  

Rubrobacter radiotolerans is the most radioresistant bacterium showing 16 kGy as D-37 against gamma-rays. However mechanisms of the radioresistance have been still unclear. To clarify the post-irradiating events in the cell, we investigated DNA strand breaks which is the most major lesion induced by ionizing radiation. The neutral sucrose density gradient centrifugation analysis showed that size of the chromosomal DNA gradually decreased with irradiation dose. However, the frequency of DNA strand breaks after ionizing irradiation was significantly lower than those reported for other eubacteria. The reduced DNA sizes were not recovered during the post-irradiating cultivation. These results suggest that in this organism DNA protection from damage is mainly involved in the radioresistance, but not DNA repair activities.

DOI： 10.1016/S0944-5013(99)80011-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN RADIATION RESEARCH SOC  

Halobacterium salinarium, a member of the extremely halophilic archaebacteria, contains a C-50-carotenoid namely bacterioruberin. We have previously reported the high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV). In this study, we have examined whether bacterioruberin and the highly concentrated salts in this bacterium play protective roles against the lethal actions of ionizing radiation, UV, hydrogen peroxide, and mitomycin-C (MMC).
The colourless mutant of H. salinarium deficient in bacterioruberin was more sensitive than the red-pigmented wild-type to all tested DNA-damaging agents except MMC. Circular dichroism (CD) spectra of H. salinarium chromosomal DNA at various concentrations of KCl (0-3.5 M) were similar to that of B-DNA, indicating that no conformational changes occurred as a result of high salt concentrations. However, DNA strand-breaks induced by ionizing radiation were significantly reduced by the presence of either bacterioruberin or concentrated KCI, presumably due to scavenging of free radicals.
These results suggest that bacterioruberin and intracellular KCl of H. salinarium protect this organism against the lethal effects of oxidative DNA-damaging agents.

DOI： 10.1269/jrr.39.251

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARCEL DEKKER INC  

Reactivities of 5-formyl-2'-deoxyuridine (fdU) and its 5'-monophosphate (fdUMP) to amino acids, amines and thiol compounds in neutral aqueous solution have been studied to elucidate the postmodification of the 5 formyluracil (fU) moiety in cells. fdU and fdUMP specifically reacted with cysteine and its analogs to form thiazolidine derivatives. The reaction involved condensation of the formyl group of fU with both alpha-NH2 (or NH2 at the equevalent position) and SH groups of cysteine derivatives.

DOI： 10.1080/07328319808005164

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have recently developed a novel method for detection and quantitation of abasic (AP) sites in DNA, in which the biotinylated reagent, called the aldehyde reactive probe (ARP) specifically reacts with aldehyde groups of AP sites and biotin-tagged damage is detected by an ELISA-like assay. The present study has been carried out to improve the feasibility and the sensitivity of ARP assay. For immobilization of DNA, a protamine sulfate-coated plate was used instead of the conventional UV-irradiated plate to enhance DNA binding. As the result. the time for immobilization was shortened to 1 h without any loss of signal. The amount of [H-3]-labeled DNA bound to the plate was proportional to the DNA concentration employed. When DNA containing AP sites was treated with ARP in solution prior to coating the protamine-plate, the sensitivity of the assay was greatly increased. A linear relationship between the DNA concentration and the signal intensity was also observed. Thus, similar to 0.1 fmol of AP sites (0.5 sites per 10(5) nt) could be detected in DNA isolated from HeLa cells after treatment with a sublethal dose (0.5 mM) of methylmethanesulfonate (MMS). Using this system, the number of total methylpurines generated by MMS in the cellular DNA was estimated after heat treatment, which converted methylated base lesions to AP sites. It was shown that the number of AP sites was about 140 sites per 10(4) nt with 25 mM MMS and 10% of total methylated bases were already released without heat depurination. (C) 1998 Elsevier Science B.V.

DOI： 10.1016/S0003-2670(97)00648-X

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Gamma-irradiation of aqueous solutions containing DNA and alcohols is known to generate fluorescent products. In this study, aqueous solutions containing 2'-deoxyguanosine (dG) and amino acids with ar. aliphatic hydroxyl group [serine (Ser) or threonine (Thr)] were irradiated and analyzed for fluorescence. Both irradiated solutions showed a similar fluorescence profile with the excitation maximum similar to 310 nm and the emission maximum similar to 370 nm. The fluorescence profiles were very close to that of 2-aminopurine, known as a highly fluorescent base, In the irradiation of dG with Ser, two distinct fluorescent products were observed in liquid chromatographic analysis. The major product was further purified by chromatography. Mass spectrometry (MS) showed that the major fluorescent product was a dG adduct bearing a Ser fragment at the C-6 position, In the case of dG with Thr, only one major fluorescent product was observed. The purified product seemed to be a C-6 adduct of dG with a Thr fragment based on MS and NMR analyses. These structural data for the fluorescent products suggest that C-6 adduct formation of dG was the key to generating fluorescence. This is consistent with our previous finding that the adduct between dG and tert-butanol at C-6 is highly fluorescent. (C) 1998 Elsevier Science B,V.

DOI： 10.1016/S0003-2670(97)00624-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD  

Endonuclease III (endoIII; nth gene product) of Escherichia coli is known to be a DNA repair enzyme having a relatively broad specificity for damaged pyrimidine bases of DNA. Here, we describe the cloning and characterization of the cDNA and the gene for a mouse homologue (mNthl1/mNth1) of endoIII. The cDNA was cloned from a mouse T-cell cDNA library with a probe prepared by PCR using the library and specific PCR primers synthesized based on the reported information of partial amino acid sequences of bovine NTHL1/NTH1 and of EST Data Bases. The cDNA is 1025 nucleotides long and encodes a protein consisting of 300 amino acids with a predicted molecular mass of 33.6 kDa. The amino acid sequence exhibits significant homologies to those of endoIII and its prokaryotic and eukaryotic homologues. The recombinant mNthl1 with a hexahistidine tag was overexpressed in a nth::cm(r) nei::Km(r) double mutant of E. coli, and purified to apparent homogeneity. The enzyme showed thymine glycol DNA glycosylase, urea DNA glycosylase and AP lyase activities. Northern blot analysis indicated that mNthl1 mRNA is about 1 kb and is expressed ubiquitously. A 15 kb DNA fragment containing the mNthl1 gene was cloned from a mouse genomic library and sequenced. The gene consists of six exons and five introns spanning 6.09 kb. The sequenced 5' flanking region lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors such as Ets, Spl, AP-1 and AP-2. The mNthl1 gene was shown to lie immediately adjacent to the tuberous sclerosis 2 (Tsc2) gene in a 5'-to-5' orientation by sequence analysis and was assigned to chromosome 17A3 by in situ hybridization. (C) 1998 Academic Press.

DOI： 10.1006/jmbi.1998.2042

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The nearly complete sequence of the 16S rRNA gene of an extremely highly radiotolerant bacterium, Rubrobacter radiotolerans (reclassified from Arthrobacter radiotolerans based on chemical characteristics), was determined by PCR amplification of the genomic DNA followed by cloning of the amplified gene and sequencing by the dideoxynucleotide method. The sequence was aligned with the sequences of members of the genus Arthrobacter and also with the sequences of representatives of the gram-positive bacteria having high G+C contents and the family Deinococcaceae (radioresistant micrococci and their relatives), The results of our phylogenetic analysis confirmed that R. radiotolerans is not a member of the Arthrobacter group and thus supported the previous reclassification. Moreover, although it is radioresistant and has a high GS-C content, R. radiotolerans is more closely related to the gram-positive bacteria with high G+C contents than to the radioresistant members of the Deinococcaceae.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, pK(a) of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (pK(a) = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5' exonuclease free) and Taq DMA polymerase, fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much Bower efficiency than that for dTTP, The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0, The parallel correlation between ionization of the base unit of fdUTP (pK(a) = 8.6) and the Substitution efficiency for dCTP strongly suggests that the base-ionized form of dFdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. in addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.

DOI： 10.1093/nar/25.8.1570

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN RADIATION RESEARCH SOC  

Lethal effects of Co-60 gamma-rays, UV light, and mitomycin C on two kinds of bacteria, Halobacterium salinarium which grows in highly concentrated salt media and Thiobacillus intermedius which requires reduced sulfur compounds, were studied and compared with those on Escherichia coli B/r. D-37 values for H. salinarium, T. intermedius and E. coli B/r were 393, 150, and 92 Gy, respectively, by exposure to Co-60 gamma-rays. They were 212, 38, and 10 J/m(2), respectively, by exposure to UV light and 2.36, 0.25, and 0.53 mu g/ml/h, respectively, by exposure to mitomycin C. Against these agents, H. salinarium was much more resistant than T. intermedius and E. coli B/r.

DOI： 10.1269/jrr.38.37

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alpha-2'-Deoxyadenosine (alpha) is a major adenine lesion produced by gamma-ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing alpha were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for a were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site. The transfection assay revealed that alpha constituted a moderate block to DNA replication. The in vivo replication capacity to pass through alpha was similar to 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block. Similar data were obtained by in vitro replication of oligonucleotide templates containing alpha or F by E. coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing alpha was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of alpha introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated alpha was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G. A possible mechanism of the single nucleotide deletion associated with alpha is discussed on the basis of the misinsertion-strand slippage model.

DOI： 10.1093/nar/25.3.597

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD LONDON  

Previously we reported haematopoietic death as an effect of tritiated water (HTO) in drinking water in the concentration range from 5 . 92 X 10(11) to 1 . 85 X 10(10) Bq/dm(3). In the present study the effects of HTO in a lower concentration range from 9 . 25 X 10(9) Bq/dm(3) (0 . 240 Gy/day) to 3 . 70 X 10(8) Bq/dm(3) (0 . 096 Gy/day) are reported. Female (C57BL/6N and C3H/He)F-1 mice were maintained on drinking water containing various levels of HTO. Mice survived for &gt; 150 days with a high incidence of tumour development (70 to 80%). In the dose-rate range from 9 . 25 X 10(9) Bq/dm(3) (0 . 240 Gy/day) to 1 . 85 X 10(9) Bq/dm(3) (0 . 048 Gy/day) the main cause of death was thymic lymphoma. However, at a dose-rate of 9 . 25 X 10(8) Bq/dm(3) (0 . 024 Gy/day) the incidence of thymic lymphoma sharply decreased, while the incidence of other tumours increased. The tumour types became more diverse at lower concentrations of HTO. The latent period of tumour development was shorter and the life-shortening effect was more marked by H-3 beta-irradiation in this study than by X- or gamma-irradiation reported in other investigations.

DOI： 10.1080/09553009514550911

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

2'-Deoxyguanosine in aqueous solution (5 x 10(-4) mol/dm3) was irradiated with Co-60 gammarays in the presence of t-BuOH (10(-1) mol/dm3) under N2. A very highly fluorescent product was isolated by gel chromatography (Cellulofine GC-15-m) and high performance liquid chromatography (Supelcosil LC-8-DB). A longer wavelength shift of absorption maxima in UV spectrum and no C=O stretching absorption in IR spectrum as compared to the original compound were found. The mass spectra of the product and its TMS derivative suggested that the very highly fluorescent product was 2-amino-6-(t-hydroxybutyl)-9-(2'-deoxyribosyl)-purine. This was confirmed by measurements of H-1 and C-13 NMR and also by elemental analysis. The production yield, G value, was 0.1. The addition of alcohol radical and the elimination of OH group at the C-6 position of guanine base ring is a new finding and an interesting reaction with its very highly fluorescent nature. Therefore, this reaction is important radiochemically rather than radiobiologically.

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2′-Deoxyguanosine in aqueous solution (5 × 10-4 mol/dm3) was irradiated with 60Co gamma-rays in the presence of t-BuOH (10-1 mol/dm3) under N2. A very highly fluorescent product was isolated by gel chromatography (Cellulofine GC-15-m) and high performance liquid chromatography (Supelcosil LC-8-DB). A longer wavelength shift of absorption maxima in UV spectrum and no C=O stretching absorption in IR spectrum as compared to the original compound were found. The mass spectra of the product and its TMS derivative suggested that the very highly fluorescent product was 2-amino-6-(t-hydroxybutyl)-9-(2′-deoxyribosyl)-purine. This was confirmed by measurements of 1H and 13C NMR and also by elemental analysis. The production yield, G value, was 0.1. The addition of alcohol radical and the elimination of OH group at the C-6 position of guanine base ring is a new finding and an interesting reaction with its very highly fluorescent nature. Therefore, this reaction is important radiochemically rather than radiobiologically. © 1994.

DOI： 10.1016/0969-806X(94)00062-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The highly radioresistant Rubrobacter radio-tolerans,, contains red pigments. Since the pigments could not be extracted by usual methods, a new method was developed in which the pigments were extracted with organic solvents after addition of 10 N KOH to the intact cells, followed by neutralization. These pigments were also extracted after treatment with achromopeptidase, but not with lysozyme, The extracted pigments separated into two main spots by TLC (48.6% and 22.6%), and were confirmed to be carotenoids by chemical tests. The two major pigments had 13 conjugated double bonds as determined from the main maximum wavelength of the light absorption spectra. Their molecular weights were determined to be 740 and 722 by mass spectrometry. The mass spectra of their TMS-derivatives revealed that they contained four and three tertiary OH groups, respectively. Confirming their identical light and IR spectra, these pigments were determined to be bacterioruberin and monoanhydrobacterioruberin, respectively, the characteristic carotenoids of halophilic bacteria. The existence of these pigments in bacteria other than halobacteria provides interesting new evidence on the distribution of these compounds.

DOI： 10.1007/s002030050159

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Ionizing radiation generates isolated and localized multiple (i.e., clustered) lesions in DNA, and the latter is believed to be primarily responsible for deleterious biological consequences such as cell killing and mutations. In the present study, we irradiated plasmid and lambda DNA by ionizing radiations with different linear energy transfer (LET) values, and compared the yields of isolated and clustered DNA lesions.

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Endo III and Endo VIII are major E. coli DNA glycosylases that remove oxidatively damaged pyrimidine bases. In the present study, we have compared the damage specificity of human homologues of Endo III (hNTHl) and Endo VIII (hNEIL1 and hNEIL2) to elucidate the repair role in cells. hNTH1 and hNEIL1 recognized a similar spectra of bases lesions, but the preference of damage including the stereoisomers of thymine glycol was significantly different between hNTH1 and hNEIL1. hNEIL2 exhibited a strong AP lyase activity but the N-glycosylase activity for the tested oxidative base lesions was marginal.

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Single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1) was previously identified as a putative backup enzyme of major mammalian uracil-DNA glycosylase (UDG). However, the subsequent studies have shown conflicting results about the substrate specificity of SMUG1. In the present study, to clarify the repair role of SMUG1, we determined the damage specificity of purified human SMUG1 (hSMUG1) and its contribution to repair of oxidized bases in HeLa cell extracts.

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5-Formyluracil is a major oxidative thymine lesion with mutagenic and cytotoxic properties. In this study, we have partially purified and characterized a mammalian 5-formyluracil-DNA glycosylase (FDG) from rat liver. FDG was a monofunctional DNA glycosylase and removed 5-formyluracil, uracil, 5-hydroxyuracil, 5-hydroxylmethyluracil in single-stranded and double-stranded DNA. Several lines of evidence indicate that FDG is a rat SMUG1 homologue. Human SMUG1 also exhibited similar enzymatic properties.

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5-Hydroxyuracil (HOU) and 5-hydroxycytosine (HOC) are major oxidative lesions of cytosine with mutagenic potentials. Therefore, HOU and HOC need to be removed from DNA to avoid mutation. In this study, oligonucleotide substrates containing HOU and HOC were synthesized by DNA polymerase reactions and tested for DNA glycosylases. Ung exhibited an extremely low activity for HOU as compared to uracil (U). In contrast, hSMUG1 excised HOU and U with a comparable efficiency. Ung and hSMUG1 did not excise HOC.

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When DNA is exposed to NO or HNO2, oxanine (Oxa) is formed as a major guanine lesion. For highly sensitive detection of Oxa using ARP, a probe molecule for DNA damage detection, the reactivity of ARP to Oxa was examined. Oxa site-specifically embedded in an oligonucleotide reacted with ARP but it took relatively long time (ca. 24 h) to completely convert Oxa to an ARP-labeled form.

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DOI： 10.1093/nass/1.1.47

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DOI： 10.1093/nass/1.1.45

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Halobacterium salinarium is an extremely halophilic archaebacterium, which lives in the surface of salt lake and salt plant. In these environments, H. salinarium is usually exposed by intense irradiation of sunlight including ultraviolet light (UV). UV produces DNA lesions, and possesses mutagenic and/or lethal effects on organisms. H. salinarium can tolerant various DNA-damaging agents such as UV and gamma rays. H. salinarium contains saturated concentrations of K+ and Cl- in the cell. Since Cl- is known to be a radical scavenger, the intracellular highly accumulated ions seem to be involved in the DNA protection. In vitro, highly concentrated KCI inhibited the generations of cyclobutane pyrimidine dimer (CPD) and DNA strand break, the DNA lesions induced by UV and gamma rays, respectively. These protection efficiencies are larger than that shown by carotenoid, an abandon antioxidant in the cell of H. salinarium. Therefore the intracellular highly concentrated salts play effective role not only to tolerant against osmotic pressure, but also to maintain the genetic information in the extremely severe environment.

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