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E. coli YdbK is predicted to be a pyruvate:flavodoxin oxidoreductase (PFOR). However, enzymatic activity and the regulation of gene expression of it are not well understood. In this study, we found that E. coli cells overexpressing the ydbK gene had enhanced PFOR activity, indicating the product of ydbK to be a PFOR. The PFOR was labile to oxygen. The expression of ydbK was induced by superoxide generators such as methyl viologen (MV) in a SoxS-dependent manner after a lag period. We identified a critical element upstream of ydbK gene required for the induction by MV and proved direct binding of SoxS to the element. E. coli ydbK mutant was highly sensitive to MV, which was enhanced by additional inactivation of fpr gene encoding ferredoxin (flavodoxin):NADP(H) reductase (FPR). Aconitase activity, a superoxide sensor, was more extensively decreased by MV in the E. coli ydbK mutant than in wild-type strain. The induction level of soxS gene was higher in E. coli ydbK fpr double mutant than in wild-type strain. These results indicate that YdbK helps to protect cells from oxidative stress. It is possible that YdbK maintains the cellular redox state together with FPR and is involved in the reduction of oxidized proteins including SoxR in the late stages of the oxidative stress response in E. coli.

Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSoxTM Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2', 7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47, 000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation.

Escherichia coliDNA polymerase IV (Pol IV) is involved in bypass replication of damaged bases in DNA. Reactive oxygen species (ROS) are generated continuously during normal metabolism and as a result of exogenous stress such as ionizing radiation. ROS induce various kinds of base damage in DNA. It is important to examine whether Pol IV is able to bypass oxidatively damaged bases. In this study, recombinant Pol IV was incubated with oligonucleotides containing thymine glycol (dTg), 5-formyluracil (5-fodU), 5-hydroxymethyluracil (5-hmdU), 7,8-dihydro-8-oxoguanine (8-oxodG) and 1,2-dihydro-2-oxoadenine (2-oxodA). Primer extension assays revealed that Pol IV preferred to insert dATP opposite 5-fodU and 5-hmdU, while it inefficiently inserted nucleotides opposite dTg. Pol IV inserted dCTP and dATP opposite 8-oxodG, while the ability was low. It inserted dCTP more effectively than dTTP opposite 2-oxodA. Pol IV's ability to bypass these lesions decreased in the order: 2-oxodA > 5-fodU~5-hmdU > 8-oxodG > dTg. The fact that Pol IV preferred to insert dCTP opposite 2-oxodA suggests the mutagenic potential of 2-oxodA leading to A:TG:C transitions. Hydrogen peroxide caused an ~2-fold increase in A:TG:C mutations inE. coli, while the increase was significantly greater inE. colioverexpressing Pol IV. These results indicate that Pol IV may be involved in ROS-enhanced A:TG:C mutations.

The 5-formyluracil (5-foU), a major mutagenic oxidative damage of thymine, is removed from DNA by Nth, Nei and MutM in Escherichia coli. However, DNA polymerases can also replicate past the 5-foU by incorporating C and G opposite the lesion, although the mechanism of correction of the incorporated bases is still unknown. In this study, using a borohydride-trapping assay, we identified a protein trapped by a 5-foU/C-containing oligonucleotide in an extract from E. coli mutM nth nei mutant. The protein was subsequently purified from the E. coli mutM nth nei mutant and was identified as KsgA, a 16S rRNA adenine methyltransferase. Recombinant KsgA also formed the trapped complex with 5-foU/C- and thymine glycol (Tg)/C-containing oligonucleotides. Furthermore, KsgA excised C opposite 5-foU, Tg and 5-hydroxymethyluracil (5-hmU) from duplex oligonucleotides via a beta-elimination reaction, whereas it could not remove the damaged base. In contrast, KsgA did not remove C opposite normal bases, 7,8-dihydro-8-oxoguanine and 2-hydroxyadenine. Finally, the introduction of the ksgA mutation increased spontaneous mutations in E. coli mutM mutY and nth nei mutants. These results demonstrate that KsgA has a novel DNA glycosylase/AP lyase activity for C mispaired with oxidized T that prevents the formation of mutations, which is in addition to its known rRNA adenine methyltransferase activity essential for ribosome biogenesis.

Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei-like proteins, NEIL1 and NEIL2. A third Nei-like protein, NEIL3, is composed of an amino-terminal Nei-like domain and an unknown carboxy-terminal domain. In contrast to the other well-described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1-290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1-290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single-stranded (ss) DNA but double-stranded (ds) DNA. The activity is abrogated completely in mutants with an amino-terminal deletion and at the zinc-finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.

Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO(3)-inducing cytosine deamination than wild-type strain.

Bacteria and eukaryotes possess redundant enzymes that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases remove damaged bases to initiate the base excision repair. The exocyclic methyl group of thymine does not escape oxidative damage to produce 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). 5-foU is a potentially mutagenic lesion. A homolog of E. coli endonuclease III (SpNth1) had been identified and characterized in Schizosaccharomyces pombe. In this study, we found that SpNth1 recognizes and removes 5-foU and 5-hmU from DNA with similar efficiency. The specific activities for the removal of 5-foU and 5-hmU were comparable with that for thymine glycol. The expression of SpNth1 reduced the hydrogen peroxide toxicity and the frequency of spontaneous mutations in E. coli nth nei mutant. It was also revealed that SpNth1 had DNA glycosylase activity for removing 8-oxo-7,8-dihydroguanine (8-oxoG) from 8-oxoG/G and 8-oxoG/A mispairs. These results indicated that SpNth1 has a broad substrate specificity and is involved in the base excision repair of 8-oxoG and thymine residues oxidized in the methyl group in S. pombe.

It is of interest to perform a systematic comparative analysis of the conserved domains in DNA glycosylases and the evolution of DNA base excision repair systems. Furthermore, it is important to characterize the roles and regulation of base excision repair during the development of organisms. To address these issues, we first identified 8-oxo-7,8-dihydroguanine (8-oxoG)-DNA glycosylase (Ogg1) of the ascidian Ciona intestinalis as a good model system.A cDNA clone coding for a peptide with homology to human Ogg1 was identified in the expressed sequence tag (EST) database from the Ciona cDNA resources. We examined whether CiOgg1 has DNA glycosylase/AP (apurinic/apyrimidinic) lyase activities for 8-oxoG-containing oligonucleotide. Furthermore, the expression level of CiOgg1 was compared in various tissues of Ciona intestinalis.The CiOgg1gene encoded a protein of 351 amino acids, which shows 37% identity of amino acid sequence with human Ogg1. The Helix-hairpin-Helix motif was highly conserved. The ascidian enzyme had functional 8-oxoG-DNA glycosylase/AP lyase activities, which removed 8-oxoG opposite cytosine from DNA. Expression of the CiOgg1 significantly reduced the frequency of spontaneous G:C to T:A transversions in E. coli mutM mutY. The highest expression level was observed in testis in Ciona intestinalis.The structure and functions of Ogg1 are well conserved in Ciona intestinalis. CiOgg1 is involved in the repair of 8-oxoG in DNA in Ciona intestinalis.

Bacteria and eukaryotes possess redundant activities that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases excise damaged bases to initiate the base excision repair pathway. hOgg1 and hNTH1, homologues of E. coli MutM and Nth, respectively, had been identified and characterized in human cells. Recent works revealed that human cells have three orthologues of E. coli Nei, hNEIL1, hNEIL2 and hNEIL3. In the present experiments, hNEIL1 protected the E. coli nth nei mutant from lethal effect of hydrogen peroxide and high frequency of spontaneous mutations under aerobic conditions. Furthermore, hNEIL1 efficiently cleaved double stranded oligonucleotides containing 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU) in vitro via beta- and delta-elimination reactions. Similar activities were detected with hNTH1. These results indicate that hNEIL1 and hNTH1 are DNA glycosylases that excise 5-foU and 5-hmU as efficiently as Tg in human cells.

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The present authors reported previously that MutM, Nth and Nei in Escherichia coli removed 5-foU from DNA. The present study identified 5-foU DNA glycosylases in Saccharomyces cerevisiae in order to clarify the repair mechanisms of 5-foU in eukaryotic cells.The borohydride-trapping assay and DNA-nicking assay were carried out to detect and characterize the repair activities for 5-foU in extracts from S. cerevisiae with oligonucleotides containing 5-foU at specific sites.Two proteins in crude extracts from S. cerevisiae formed covalent complexes with oligonucleotides containing site-specific 5-foU in the presence of NaBH4. Extracts from S. cerevisiae strains defective in either the NTG1 or the NTG2 gene lacked either one or the other of these two proteins. Purified Ntg1 and Ntg2 were trapped in such complexes by the 5-foU-containing oligonucleotides in the presence of NaBH4. Furthermore, purified Ntg1 and Ntg2 efficiently cleaved the oligonucleotide at the 5-foU site.The results indicate that both Ntg1 and Ntg2 are involved in the repair of 5-foU in DNA, and thereby serve to reduce mutations in S. cerevisiae.

Mismatched or damaged base pairs in DNA are mutagenic and both eukaryotes and prokaryotes have a series of repair systems that decrease a spontaneous mutation rate. All exocyclic amino groups of cytosine(C), adenine(A), and guanine(G) contribute to hydrogen bonds for base pairing. High temperature and oxidative stresses increase the deamination of these bases and methylated C. These deaminated sites would be initially recognized by components of DNA repair system. We discovered a novel G/thymine(T)-mismatch binding protein (nGTBP) that bound, with high affinity, to a minimal 14-mer DNA heteroduplex with a strict 5'-TRT GNB-3' sequence (R for purine, N for any bases, and B for "not A," namely for C, G, or T ). This italicized G position mismatched with T could be replaced by hypoxanthine, the deaminated A. The nGTBP, however, barely recognized DNA duplexes individually containing 8-oxo-G, thymine glycol, and 5-methylcytosine.

Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei (endonuclease VIII). Tissue expression of this mouse Nei-like (designated as Neil1) gene is ubiquitous but much lower than that of mNth1 except in heart, spleen, and skeletal muscle. Recombinant NEIL1 can remove thymine glycol and 5-hydroxyuracil in double- and single-stranded DNA much more efficiently than 8-oxoguanine and can nick the strand by an associated (beta-delta) apurinic/apyrimidinic lyase activity. In addition, the mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression.

5-Formyluracil (5-foU) is a major derivative of thymine produced in DNA by ionizing radiation and various chemical oxidants. It has been previously shown that 5-foU in template DNA directs misincorporation of nucleotides by DNA polymerases during in vitro DNA synthesis. The present experiments were designed to understand the biological effects of5-foU in vivo.The modified base was incorporated site-specifically into the recognition site of restriction endonuclease SalI (5'-GTCGAC) or AflII (5'-CTTAAG) in vector plasmid pSVK3 and introduced the plasmid into Escherichia coli.When the plasmids were replicated in E. coli, 5-foU caused mutations at the target sites. The induced mutation frequencies were 0.038-0.049%. Sequence analysis revealed that 5-foU preferentially caused T:A--C:G and T:A--A:T base substitutions and -1 deletions at the 5-foU site. 5-FoU also caused mutations at sites near the 5-foU. The alkA mutation did not affect the frequency of mutations in 5-foU-containing plasmids.The present experiments demonstrated that 5-formyluracil in DNA caused mutations in E. coli.

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood. In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites. The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides. Extracts from strains defective in thenth, nei, or mutM gene lacked one of the proteins. All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant. The introduction of a plasmid carrying the nth,nei, or mutM gene into the E. colitriple mutant restored the formation of the corresponding protein-DNA complex. Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH4. Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site. In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E. coli. The mutation frequency of the plasmid was significantly increased in theE. coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains. From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli.

The post-replicative adenine:8-oxo-7,8-dihydroguanine (GO) mismatch is crucial for G:C to T:A transversion. This mismatch is corrected by Escherichia coli MutY which excises the adenine from A:GO. A candidate gene coding for the human counterpart of MutY has been cloned as hMYH. However, the function and enzyme activities of the gene product have not been identified. We previously demonstrated that an epitope-tagged hMYH protein behaves as a mitochondrial protein. In the present study, we have identified an alternative hMYH transcript, termed type 2, which differs in the exon 1 sequence of the known transcript (type 1). A nuclear localization for the type 2 protein was revealed by detection of epitope-tagged protein in COS-7 cells. Expression of both type 1 and type 2 transcripts was reduced in post-mitotic tissues. hMYH cDNA suppressed the mutator phenotype of E.coli mutY. In vitro expressed hMYH showed adenine DNA glycosylase activity toward the A:GO substrate. The protein can bind to A:GO, and to T:GO and G:GO without apparent catalysis. These results represent the first demonstration of the function of the hMYH gene product which is differentially transported into the nucleus or the mitochondria by alternative splicing

The present experiments were done to elucidate the roles of thioredoxin and thioredoxin reductase system in defense against hydrogen peroxide (H2O2) in Escherichia coli. The thioredoxin-deficient mutant (trxA) was more sensitive to H2O2 than was the wild-type strain, when challenged in the stationary and exponentially growing phase. Thioredoxin reductase-deficient mutant (trxB) in the stationary phase also exhibited increased sensitivity, compared with the wild-type strain. These results indicated that reduced form of thioredoxin is required for defense against H2O2, possibly by scavenging radicals generated in the cells. In contrast, the trxB mutant in the growing phase had higher survival after exposure to H2O2 than the wild-type strain. The acquirement of resistance related to increased capacity for removing H2O2 in the trxB mutant and was not observed in a catalase-negative background. Furthermore, enhanced expression of the katG :: lacZ gene occurred in the mutant. Therefore, it was concluded that oxidized form of thioredoxin confers H2O2 resistance on E. coli cells by increasing activity to remove H2O2, which was brought about by enhanced induction of the katG-coded catalase/hydroperoxidase I at the transcriptional level. In addition, this resistance to H2O2 correlated well with reduced amount of DNA damage caused by H2O2, determined by the induction level of the recA :: lacZ fusion gene after treatment with H2O2.

5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.

E. coli YdbK is predicted to be a pyruvate:flavodoxin oxidoreductase (PFOR). However, enzymatic activity and the regulation of gene expression of it are not well understood. In this study, we found that E. coli cells overexpressing the ydbK gene had enhanced PFOR activity, indicating the product of ydbK to be a PFOR. The PFOR was labile to oxygen. The expression of ydbK was induced by superoxide generators such as methyl viologen (MV) in a SoxS-dependent manner after a lag period. We identified a critical element upstream of ydbK gene required for the induction by MV and proved direct binding of SoxS to the element. E. coli ydbK mutant was highly sensitive to MV, which was enhanced by additional inactivation of fpr gene encoding ferredoxin (flavodoxin):NADP(H) reductase (FPR). Aconitase activity, a superoxide sensor, was more extensively decreased by MV in the E. coli ydbK mutant than in wild-type strain. The induction level of soxS gene was higher in E. coli ydbK fpr double mutant than in wild-type strain. These results indicate that YdbK helps to protect cells from oxidative stress. It is possible that YdbK maintains the cellular redox state together with FPR and is involved in the reduction of oxidized proteins including SoxR in the late stages of the oxidative stress response in E. coli.

A new method for the synthesis of 5-foU-containing oligonuleotides by phosphoramidite chemistry and subsequent post-oxidation with sodium periodate was developed. 5-(1,2-Dihydroxyethyl)uracil-containing oligomers, which are readily converted to 5-foU-containing oligomers by sodium periodate oxidation, were synthesized according to the standard β-cyanoethyl phosphoramidite chemistry. The phosphoramidite of a protected 5-(1,2-dihydroxyethyl)-2′-deoxyuridine derivative was prepared from 5-iodo-2′-deoxyuridine in 7 steps. UV melting behavior of these three oligomers demonstrated that the 5-foU-A base pair are less stable than the T-A base pair.

We examined whether or not peroxide stress induces a repair capacity for oxidatively damaged DNA in Escherichia coli cells. Peroxide stress was brought about by adding 30 microM hydrogen peroxide (H2O2) to exponentially growing cells. The following results were obtained. (1) After exposure to H2O2, E. coli resistance to X-rays was enhanced. The acquisition of resistance was inhibited by rifampicin and chloramphenicol. (2) The response was acquired in mutants defective in the katG and oxyR genes, as well as in the wild-type strain. (3) Lambda phages damaged by exposure to H2O2 showed higher survival on H2O2-treated cells than on untreated cells. (4) The peroxide stress did not render E. coli cells resistant to UV and mitomycin C. These suggest that peroxide stress induces a repair capacity against oxidative DNA damage and that this response must be regulated by a different mechanism from oxyR(+)-mediated regulatory system.

Oxidatively damaged bases in DNA cause many types of deleterious effects. The main enzyme that removes such lesions is DNA glycosylase, and accordingly, DNA glycosylase plays an important role in genome stability. Recently, a relationship between DNA glycosylases and aging has been suggested, but it remains controversial. Here, we investigated DNA glycosylases of C. elegans, which is a useful model organism for studying aging. We firstly identified a C. elegans homolog of endonuclease III (NTH), which is a well-conserved DNA glycosylase for oxidatively damaged pyrimidine bases, based on the activity and homology. Blast searching of the Wormbase database retrieved a sequence R10E4.5, highly homologous to the human NTH1. However, the R10E4.5-encoded protein did not have NTH activity, and this was considered to be due to lack of the N-terminal region crucial for the activity. Therefore, we purified the protein encoded by the sequence containing both R10E4.5 and the 117-bp region upstream from it, and found that the protein had the NTH activity. The endogenous CeNTH in the extract of C. elegans showed the same DNA glycosylase activity. Therefore, we concluded that the genuine C. elegans NTH gene is not the R10E4.5 but the sequence containing both R10E4.5 and the 117-bp upstream region. NTH-deficient C. elegans showed no difference from the wild-type in lifespan and was not more sensitive to two oxidizing agents, H2O2 and methyl viologen. This suggests that C. elegans has an alternative DNA glycosylase that repairs pyrimidine bases damaged by these agents. Indeed, DNA glycosylase activity that cleaved thymine glycol containing oligonucleotides was detected in the extract of the NTH-deficient C. elegans.

Base moieties in DNA are spontaneously threatened by naturally occurring chemical reactions such as deamination, hydrolysis and oxidation. These DNA modifications have been considered to be major causes of cell death, mutations and cancer induction in organisms. Organisms have developed the DNA base excision repair pathway as a defense mechanism to protect them from these threats. DNA glycosylases, the key enzyme in the base excision repair pathway, are highly conserved in evolution. Uracil constantly occurs in DNA. Uracil in DNA arises by spontaneous deamination of cytosine to generate pro-mutagenic U:G mispairs. Uracil in DNA is also produced by the incorporation of dUMP during DNA replication. Uracil-DNA glycosylase (UNG) acts as a major repair enzyme that protects DNA from the deleterious consequences of uracil. The first UNG activity was discovered in E. coli in 1974. This was also the first discovery of base excision repair. The sequence encoded by the ung gene demonstrates that the E. coli UNG is highly conserved in viruses, bacteria, archaea, yeast, mice and humans. In this review, we will focus on central and recent findings on the generation, biological consequences and repair mechanisms of uracil in DNA and on the biological significance of uracil-DNA glycosylase.

The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with and is functionally related to the structural maintenance of chromosomes 5 and 6 (SMC5/6) protein complex. In this study, we investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU. Significantly, Rad60 function is not required during HU arrest but is required on release. However, the mutant cells underwent aberrant mitosis accompanied by irregular segregation of chromosomes, and DNA replication was not completed, as revealed by pulsed-field gel electrophoresis. The deletion of rhp51 suppressed the aberrant mitosis of rad60-1 cells and caused mitotic arrest. These results suggest that Rhp51 and Rad60 are required for the restoration of a stalled or collapsed replication fork after release from the arrest of DNA replication by HU. The rad60-1 mutant was proficient in Rhp51 focus formation after release from the HU-induced arrest of DNA replication or DNA-damaging treatment. Furthermore, the lethality of a rad60-1 rqh1Delta double mutant was suppressed by the deletion of rhp51 or rhp57. These results suggest that Rad60 is required for recombination repair at a step downstream of Rhp51. We propose that Rhp51-dependent DNA structures that cannot activate the mitotic checkpoints accumulate in rad60-1 cells.

In order to clarify the cellular processing and repair mechanisms for radiation-induced clustered DNA damage, we examined the correlation between the levels of DNA glycosylases and the sensitivity to ionizing radiation in Escherichia coli.The lethal effects of gamma-rays, X-rays, alpha-particles and H2O2 were determined in E. coli with different levels of DNA glycosylases. The formation of double-strand breaks by post-irradiation treatment with DNA glycosylase was assayed with gamma-irradiated plasmid DNA in vitro.An E. coli mutM nth nei triple mutant was less sensitive to the lethal effect of sparsely ionizing radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of MutM (8-oxoguanine-DNA glycosylase), Nth (endonuclease III) and Nei (endonulease VIII) increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Increased sensitivity to gamma-rays also occurred in E. coli overproducing human 8-oxoguanine-DNA glycosylase (hOgg1). Treatment of gamma-irradiated plasmid DNA with purified MutM converted the covalently closed circular to the linear form of the DNA. On the other hand, overproduction of MutM conferred resistance to H2O2 on the E. coli mutM nth nei mutant.The levels of DNA glycosylases affect the sensitivity of E. coli to gamma-rays and X-rays. Excessive excision by DNA glycosylases converts nearly opposite base damage in clustered DNA damage to double-strand breaks, which are potentially lethal.

Although strong static magnetic fields (SMF) are supposed to have the potential to affect biological systems, the effects have not been evaluated sufficiently. Experiments should be performed with a powerful SMF-generating apparatus to evaluate the biological effects of SMF.An Escherichia coli mutation assay was used to assess the mutagenic effects of strong SMF. Various mutant strains of E. coli were exposed to up to 9 Tesla (T) for 24 h and the frequencies of rifampicin-resistant mutations were then determined. The expression of the soxS::lacZ fusion gene was assessed by measurement of beta-galactosidase activity.The results for survival or mutation were obtained with wild-type E. coli strain GC4468 and its derivatives defective in DNA repair enzymes or redox-regulating enzymes were all negative. On the other hand, the mutation frequency was significantly increased by the SMF exposure in soxR and sodAsodB mutants, which are defective in defence mechanisms against oxidative stress. Furthermore, the expression of superoxide-inducible soxS::lacZ fusion gene was stimulated 1.4- and 1.8-fold in E. coli when exposed to 5 and 9 T, respectively.These results indicate that strong SMF induce mutations through elevated production of intracellular superoxide radicals in E. coli.

The oxidation and deamination of 5-methylcytosine (5mC) in DNA generates a base-pair between 5-hydroxymethyluracil (5hmU) and guanine. 5hmU normally forms a base-pair with adenine. Therefore, the conversion of 5mC to 5hmU is a potential pathway for the generation of 5mC to T transitions. Mammalian cells have high levels of activity of 5hmU-DNA glycosylase, which excises 5hmU from DNA. However, glycosylases that similarly excise 5hmU have not been observed in yeast or Escherichia coli. Recently, we found that E.coli MutM, Nei and Nth have DNA glycosylase activity for 5-formyluracil, which is another type of oxidation product of the thymine methyl group. In this study, we examined whether or not E.coli MutM, Nei and Nth have also DNA glycosylase activity that acts on 5hmU in vitro. When incubated with synthetic duplex oligonucleotides containing 5hmU:G or 5hmU:A, purified MutM, Nei and Nth cleaved the 5hmU:G oligonucleotide 58, 5 and 37 times, respectively, more efficiently than the 5hmU:A oligonucleotide. In E.coli, the 5hmU-DNA glycosylase activities of MutM, Nei and Nth may play critical roles in the repair of 5hmU:G mispairs to avoid 5mC to T transitions.

5-formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The elucidation of repair mechanisms for 5-foU will yield important insights into the biological consequences of the lesion. Recently, we reported that 5-foU is recognized and removed from DNA by Escherichia coli enzymes Nth (endonuclease III), Nei (endonuclease VIII) and MutM (formamidopyrimidine DNA glycosylase). Human cells have been shown to have enzymatic activities that release 5-foU from X-ray-irradiated DNA, but the molecular identities of these activities are not yet known. In this study, we demonstrate that human hNTH1 (endonuclease III homolog) has a DNA glycosylase/AP lyase activity that recognizes 5-foU in DNA and removes it. hNTH1 cleaved 5-foU-containing duplex oligonucleotides via a beta-elimination reaction. It formed Schiff base intermediates with 5-foU-containing oligonucleotides. Furthermore, hNTH1 cleaved duplex oligonucleotides containing all of the 5-foU/N pairs (N = G, A, T or C). The specific activities of hNTH1 for cleavage of oligonucleotides containing 5-foU and thymine glycol were 0.011 and 0.045 nM/min/ng protein, respectively. These results indicate that hNTH1 has DNA glycosylase activity with the potential to recognize 5-foU in DNA and remove it in human cells.

2-Hydroxyadenine (2-ohA) is an oxidation product of adenine generated in DNA by ionizing radiation and various chemical oxidants. 2-ohA has mutational potential comparable to that of 8-oxoguanine in bacteria and mammalian cells. Recent studies have shown that 2-ohA is removed from DNA by a human MutY homolog, MYH protein, in vitro. On the other hand, the repair mechanisms for 2-ohA in Escherichia coli are not yet understood.Gel shift assays were used to assess the binding activity of E. coli full-length MutY protein and its N-terminal (residues 1-226) domain (M25) to 2-ohA/G-, 2-ohA/A-, 2-ohA/C- and 2-ohA/T-containing 24-mer oligonucleotides. Furthermore, whether these proteins specifically cleave 2-ohA-containing duplex oligonucleotides was examined.The purified MutY and M25 proteins had similar binding affinities to 2-ohA/G-, 2-ohA/A- and 2-ohA/C-containing oligonucleotides. MutY protein removed 2-ohA preferentially from 2-ohA/G mispairs. M25 protein showed the reduced catalytic activity for 2-ohA/G-containing oligonucleotides.E. coli MutY protein has a DNA glycosylase activity that removes 2-ohA from 2-ohA/G mispairs in DNA. The C-terminal domain is required for the removal of 2-ohA from DNA, but is not crucial for binding to 2-ohA-containing oligonucleotides.

The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C→C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.

The pathways leading to G:C--C:G transversions and their repair mechanisms remain uncertain. C/C and G/G mismatches arising during DNA replication are a potential source of G:C--C:G transversions. The Escherichia coli mutHLS mismatch repair pathway efficiently corrects G/G mismatches, whereas C/C mismatches are a poor substrate. Escherichia coli must have a more specific repair pathway to correct C/C mismatches. In this study, we performed gel-shift assays to identify C/C mismatch-binding proteins in cell extracts of E. COLI: By testing heteroduplex DNA (34mers) containing C/C mismatches, two specific band shifts were generated in the gels. The band shifts were due to mismatch-specific binding of proteins present in the extracts. Cell extracts of a mutant strain defective in MutM protein did not produce a low-mobility complex. Purified MutM protein bound efficiently to the C/C mismatch-containing heteroduplex to produce the low-mobility complex. The second protein, which produced a high-mobility complex with the C/C mismatches, was purified to homogeneity, and the amino acid sequence revealed that this protein was the FabA protein of E.COLI: The high-mobility complex was not formed in cell extracts of a fabA mutant. From these results it is possible that MutM and FabA proteins are components of repair pathways for C/C mismatches in E.COLI: Furthermore, we found that Saccharomyces cerevisiae OGG1 protein, a functional homolog of E.COLI: MutM protein, could specifically bind to the C/C mismatches in DNA.

Escherichia coli HU protein exists as a heterodimer composed of two highly homologous subunits, HU-1 and HU-2, encoded by the hupB and hupA genes, respectively. It introduces negative supercoils into a relaxed circular DNA. Various roles of HU have been suggested in cellular processes such as DNA replication and transcription. The present experiments were designed to understand the role of HU in DNA repair processes in E. coli.The sensitivity of hupA/hupB mutants of E. coli to the lethal and mutagenic effects of UV was compared with that of a wild-type strain. The effect of the hupAhupB mutations in SOS induction was also examined.The hupAhupB mutations increased the UV sensitivity of E. coli. Nucleotide excision repair was unaffected by the deficiency of HU. On the other hand, E. coli hupAhupB mutants were sensitive to UV in the recA+recB+recF background but not in the recArecB+recF+ or recA+recBrecF+ background. The frequency of UV-induced mutation to rifampicin resistance was significantly reduced in the hupAhupB mutants, and the induction of the recA::lacZ and umuC::lacZ fusion genes was also suppressed in the mutants.HU protein plays a critical role in the recA, recB-dependent recombinational DNA repair and SOS induction pathways in UV-irradiated E. coli.

To investigate the biological consequences of 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU).The authors constructed 22-mer oligonucleotides containing a 5-foU or 5-hmU residue at the same sites. The effects of such modifications on the ability to serve as a template for DNA polymerase and on the cleavage by sequence-specific restriction endonuclease were examined.The Klenow fragment of DNA polymerase I and Thermus thermophilus DNA polymerase read through the sites of 5-foU and 5-hmU in the templates. 5-FoU directed the incorporation of dCMP in addition to dAMP opposite the lesion during DNA synthesis. The DNA polymerases incorporated only dAMP opposite the 5-hmU. The substitution of thymine by 5-foU within the recognition site of the restriction endonucleases HincII and SalI inhibited or prevented the cleavage by the enzymes, whereas the enzymes cleaved the 5-hmU-containing oligonucleotides at the same rate as the T-containing oligonucleotides.These results indicated that the 5-foU-A base pair is less stable than the T-A base pair and that 5-foU can form a base pair with C in addition to A. It was also demonstrated that the oxidation of thymine to 5-hmU does not result in substantial deterioration.

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.

To identify a repair enzyme for 5-formyluracil (5-FU) caused by ionizing radiation in DNA, we used a radiolabelled product-release assay for this thymine-damaged substrate. Double-stranded poly(dA-dT)-poly(dA-dT) was radiolabelled by nick translation with [2-14C]-thymidine triphosphate. The DNA was irradiated with X-rays and incubated with cell extract from mouse liver. Radiolabelled products released from the irradiated DNA into an ethanol-soluble fraction were analysed by reversed-phase hplc. Released 5-FU was detected as a free base during reaction with the cell extract. 5-Formyl-2'-deoxyuridine was not detected in the ethanol supernatant. Boiling the extract at 97 degrees C for 15 min completely abolished its ability to release 5-FU. Similar enzymatic activity was observed with rat liver extract. These results demonstrated that mammalian cells have enzymatic activity to release 5-FU from DNA.

Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress.

The Escherichia coli oxyR gene is required for the induction of a regulon that is inducible by hydrogen peroxide and confers resistance to oxidative stresses. We constructed a plasmid system that greatly overproduced OxyR protein and purified the protein. OxyR protein specifically bound to the upstream regulatory regions of the oxyR and katG genes as demonstrated by the gel-retardation assay and the DNase I footprinting experiment, and activated the transcription initiation of the katG gene in vitro. Using a plasmid carrying an oxyR'-'lacZ fusion gene, we studied the regulation of oxyR expression in vivo. The expression of oxyR was not induced by the treatment with a low concentration of hydrogen peroxide which induces the genes in the oxyR regulon. The expression of the oxyR'-'lacZ gene was higher in an oxyR-deletion strain than in the oxyR+ strain, and was repressed by overexpressing the OxyR protein. These results suggest that OxyR protein functions as a repressor for oxyR, in addition to its known function as a transcriptional activator for the genes in the oxyR regulon.

We investigated the effects of 6- and 10-T static magnetic fields (SMFs) on the expression of protooncogenes using Western blot immunohybridization methods. We used a SMF exposure system, which can expose cells to a spatially inhomogeneous 6 T with a strong magnetic field (MF) gradient (41.7 T/m) and a spatially homogeneous 10 T of the highest magnetic flux density in this experiment. HL-60 cells exposed to either 6- or 10-T SMF for periods of 1 to 48 h did not exhibit remarkable differences in levels of c-Myc and c-Fos protein expression, as compared with sham-exposed cells. In contrast, c-Jun protein expression increased in HL-60 cells after exposure to 6-T SMF for 24, 36, 48, and 72 h. These results suggest that a homogeneous 10-T SMF does not alter the expression of the c-jun, c-fos, and c-myc protooncogenes. However, our observation that exposure to a strong MF gradient induced c-Jun expression suggests that a strong MF gradient may have significant biological effects, particularly regarding processes related to an elevation of c-jun gene expression.

The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.

Experiments were carried out to examine mutation induction in E. coli cells incubated in the reaction mixture of NADPH-dependent lipid peroxidation of microsomes isolated from rat liver. The results obtained were as follows: (1) Lipid peroxidation of microsomes occurred extensively on incubation with NADPH and Fe 2+ . In the E. coli WP2 uvrA (pKM101) system, the mutation frequency to streptomycin resistance increased markedly when the cells were incuated in the reaction mixture of microsomal lipid peroxidation. The induced mutation frequencies were dependent on the extent of the lipid peroxidation. (2) It was also found that the mutations were induced at the same rate as in the case (1) when the cells added to the microsomal suspensions after the reactions due to short-lived free radicals had terminated.(3) The cytotoxicity of the lipid peroxidation products was larger in the DNA repair-defective mutant, E. coli SR18 ( uvrArecA ) than the wild-type strain, SR749. From these results it is concluded that some DNA-damaging and mutagenic substances are indeed produced in the degradation process of peroxidized polyunsaturated fatty acids in liver microsomal lipids.

The katEkatG mutant of E. coli , UM1, had no assayable catalase activities in the extract and showed increased (about 20 fold) sensitivity to killing by H2O2 when compared with its parental strain CSH7. The mutant strain was able to reactivate H2O2-damaged λ phage. On the other hand, recA and polA mutants were also highly sensitive to H2O2, but they had normal level of catalase activities. RecA derivatives of UM1 were much more sensitive to H2O2 than UM1 and recA strains. The induction of umu operon occurred in UM1 at lower ( 1 10 – 1 20 ) doses of H2O2 than in CSH7. From the results it is concluded that the lethal effect of H2O2 is due to DNA damage induced by it and that catalase and DNA repair systems have a distinct role in protection against H2O2 in E. coli .