The H2TH-like motif of the Escherichia coli multifunctional protein KsgA is required for DNA binding involved in DNA repair and the suppression of mutation frequencies.

BACKGROUND: DNA oxidatively damaged by reactive oxygen species is repaired by base excision repair (BER) pathway proteins, with DNA glycosylases removing damaged or mismatched bases in the first step of BER. KsgA is a multifunctional protein that exhibits the activities of two enzymes, DNA glycosylase and rRNA dimethyltransferase. The structure-function relationship of the KsgA protein in cellular DNA repair remains unclear because the domains required for KsgA to recognize DNA have not been identified. PURPOSE: To clarify the mechanisms by which KsgA recognizes damaged DNA and to identify the DNA-binding site, which exists in KsgA. METHODS: A structural analysis and in vitro DNA-protein binding assay were performed. The C-terminal function of the KsgA protein was investigated in vitro and in vivo. RESULTS: The 3D conformations of KsgA, MutM, and Nei were compared at UCSF Chimera. The root mean square deviation of KsgA (214-273) and MutM (148-212) and that of KsgA (214-273) and Nei (145-212) were 1.067 and 1.188 Å, both less than 2 Å, suggesting that the C terminal of KsgA is spatially similar to the H2TH domains of MutM and Nei. The full-length KsgA protein and KsgA lacking 1-8 or 214-273 amino acids were purified and used in gel mobility shift assays. KsgA exhibited DNA-binding activity, which was lost in the C-terminally deleted KsgA protein. Spontaneous mutation frequency was measured using a mutM mutY ksgA-deficient strain, and the results obtained showed that the mutation frequency was not suppressed by KsgA lacking the C-terminal region, whereas it was in KsgA. To assess dimethyltransferase activity, kasugamycin sensitivity was assessed in wild-type and ksgA-deficient strains. Plasmids carrying the full-length ksgA gene and C-terminal deletion gene were introduced into ksgA-deficient strains. KsgA lacking the C terminus restored dimethyltransferase activity in the ksgA-deficient strain as well as KsgA. CONCLUSION: The present results confirmed that one enzyme exhibited two activities and revealed that the C-terminal (214-273) amino acids of KsgA were highly similar to the H2TH structural domain, exhibited DNA-binding activity, and inhibited spontaneous mutations. This site is not essential for dimethyltransferase activity.

High-Dose Irradiation Inhibits Motility and Induces Autophagy in Caenorhabditis elegans.

Radiation damages many cellular components and disrupts cellular functions, and was previously reported to impair locomotion in the model organism Caenorhabditis elegans. However, the response to even higher doses is not clear. First, to investigate the effects of high-dose radiation on the locomotion of C. elegans, we investigated the dose range that reduces whole-body locomotion or leads to death. Irradiation was performed in the range of 0-6 kGy. In the crawling analysis, motility decreased after irradiation in a dose-dependent manner. Exposure to 6 kGy of radiation affected crawling on agar immediately and caused the complete loss of motility. Both γ-rays and carbon-ion beams significantly reduced crawling motility at 3 kGy. Next, swimming in buffer was measured as a motility index to assess the response over time after irradiation and motility similarly decreased. However, swimming partially recovered 6 h after irradiation with 3 kGy of γ-rays. To examine the possibility of a recovery mechanism, in situ GFP reporter assay of the autophagy-related gene lgg-1 was performed. The fluorescence intensity was stronger in the anterior half of the body 7 h after irradiation with 3 kGy of γ-rays. GFP::LGG-1 induction was observed in the pharynx, neurons along the body, and the intestine. Furthermore, worms were exposed to region-specific radiation with carbon-ion microbeams and the trajectory of crawling was measured by image processing. Motility was lower after anterior-half body irradiation than after posterior-half body irradiation. This further supported that the anterior half of the body is important in the locomotory response to radiation.

Oxidation resistance 1 functions in the maintenance of cellular survival and genome stability in response to oxidative stress-independent DNA damage.

BACKGROUND: DNA damage is generated by various intrinsic and extrinsic sources such as reactive oxygen species (ROS) and environmental mutagens, and causes genomic alterations. DNA damage response (DDR) is activated to induce cell cycle arrest and DNA repair. Oxidation resistance 1 (OXR1) is a protein that defends cells against oxidative stress. We previously reported that OXR1 protein functions in the regulation of G2-phase cell cycle arrest in cells irradiated with gamma-rays, suggesting that OXR1 directly responds to DNA damage. PURPOSE: To clarify the functions of OXR1 against ROS-independent DNA damage, HeLa and OXR1-depleted HeLa cells were treated with heavy-ion beams and the ROS-independent DNA-damaging agent methyl methanesulfonate (MMS). RESULTS: First, OXR1-depleted cells exhibited higher sensitivity to MMS and heavy-ion beams than control cells. Next, OXR1 depletion increased micronucleus formation and shortened the duration of G2-phase arrest after treatment with MMS or heavy-ion beams. These results suggest that OXR1 functions in the maintenance of cell survival and genome stability in response to DNA damage. Furthermore, the OXR1 protein level was increased by MMS and heavy-ion beams in HeLa cells. CONCLUSIONS: Together with our previous study, the present study suggests that OXR1 plays an important role in the response to DNA damage, not only when DNA damage is generated by ROS.

The Base Excision Repair Pathway in the Nematode Caenorhabditis elegans.

Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

Deficiency of Grx1 leads to high sensitivity of HeLaS3 cells to oxidative stress via excessive accumulation of intracellular oxidants including ROS.

Oxidative stress is often initiated by excess reactive oxygen species (ROS) production, resulting in macromolecular damage, which is implicated in many disease states. Glutaredoxin 1 (Grx1) is an antioxidant enzyme that plays an important role in redox signaling and redox homeostasis. In the present study, we generated HeLaS3 cell lines deficient in Grx1 by the CRISPR/CAS9 system to clarify how Grx1 affects the physiological activities of HeLaS3 cells to respond to oxidative stress. First, the survival assay revealed that Grx1-deficient HeLaS3 cells were more sensitive to γ-ray irradiation, heat shock and H2O2 exposure than HeLaS3 wild-type cells. Next, the intracellular redox state was investigated using a fluorescent probe (2'-7'dichlorofluorescin diacetate), and the oxidized state of total proteins and a peroxidase Prx2 were measured by Western blot analysis. Exposure to γ-ray irradiation, heat shock and H2O2 significantly induced more accumulation of intracellular oxidants including ROS and higher levels of oxidized proteins in Grx1-deficient HeLaS3 cells. Furthermore, MitoSox Red staining demonstrated that Grx1 deficiency causes a higher level of oxidants production in mitochondria. Moreover, Grx1-deficient HeLaS3 cells had a higher cytochrome c level and higher apoptosis rate (Annexin-V/FITC and EthD-III staining assay) upon oxidative stress. These results suggested that Grx1 deficiency lead to mitochondrial redox homeostasis disruption and apoptotic cell death upon oxidative stress. In addition, the results of proliferation assay and MitoTracker staining assay (multinuclear cell formation rate) suggested that oxidative stress exposure inhibits cell proliferation maybe by affecting cytoplasmic division in Grx1-deficient HeLaS3 cells.

Oxidation resistance 1 prevents genome instability through maintenance of G2/M arrest in gamma-ray-irradiated cells.

Human oxidation resistance 1 (OXR1) was identified as a protein that decreases genomic mutations in Escherichia coli caused by oxidative DNA damage. However, the mechanism by which OXR1 defends against genome instability has not been elucidated. To clarify how OXR1 maintains genome stability, the effects of OXR1-depletion on genome stability were investigated in OXR1-depleted HeLa cells using gamma-rays (γ-rays). The OXR1-depleted cells had higher levels of superoxide and micronucleus (MN) formation than control cells after irradiation. OXR1-overexpression alleviated the increases in reactive oxygen species (ROS) level and MN formation after irradiation. The increased MN formation in irradiated OXR1-depleted cells was partially attenuated by the ROS inhibitor N-acetyl-L-cysteine, suggesting that OXR1-depeletion increases ROS-dependent genome instability. We also found that OXR1-depletion shortened the duration of γ-ray-induced G2/M arrest. In the presence of the cell cycle checkpoint inhibitor caffeine, the level of MN formed after irradiation was similar between control and OXR1-depleted cells, demonstrating that OXR1-depletion accelerates MN formation through abrogation of G2/M arrest. In OXR1-depleted cells, the level of cyclin D1 protein expression was increased. Here we report that OXR1 prevents genome instability by cell cycle regulation as well as oxidative stress defense.

CiApex1 has AP endonuclease activity and abrogated AP site repair disrupts early embryonic development in Ciona intestinalis.

Apurinic/apyrimidinic (AP) sites are the most common form of cytotoxic DNA damage. Since AP sites inhibit DNA replication and transcription, repairing them is critical for cell growth. However, the significance of repairing AP sites during early embryonic development has not yet been clearly determined. Here, we focused on APEX1 from the ascidian Ciona intestinalis (CiApex1), a homolog of human AP endonuclease 1 (APEX1), and examined its role in early embryonic development. Recombinant CiApex1 protein complemented the drug sensitivities of an AP endonuclease-deficient Escherichia coli mutant, and exhibited Mg2+-dependent AP endonuclease activity, like human APEX1, in vitro. Next, the effects of abnormal AP site repair on embryonic development were investigated. Treatment with methyl methanesulfonate, which alkylates DNA bases and generates AP sites, induced abnormal embryonic development. This abnormal phenotype was also caused by treatment with methoxyamine, which inhibits AP endonuclease activity. Furthermore, we constructed dominant-negative CiApex1, which inhibits CiApex1 action, and found that its expression impaired embryonic growth. These results suggested that AP site repair is essential for embryonic development and CiApex1 plays an important role in AP site repair during early embryonic development in C. intestinalis.

AP endonuclease EXO-3 deficiency causes developmental delay and abnormal vulval organogenesis, Pvl, through DNA glycosylase-initiated checkpoint activation in Caenorhabditis elegans.

AP endonuclease deficiency causes cell death and embryonic lethality in mammals. However, the physiological roles of AP endonucleases in multicellular organisms remain unclear, especially after embryogenesis. Here, we report novel physiological roles of the AP endonuclease EXO-3 from larval to adult stages in Caenorhabditis elegans, and elucidated the mechanism of the observed phenotypes due to EXO-3 deficiency. The exo-3 mutants exhibited developmental delay, whereas the apn-1 mutants did not. The delay depended on the DNA glycosylase NTH-1 and checkpoint kinase CHK-2. The exo-3 mutants had further developmental delay when treated with AP site-generating agents such as methyl methane sulfonate and sodium bisulfite. The further delay due to sodium bisulfite was dependent on the DNA glycosylase UNG-1. The exo-3 mutants also demonstrated an increase in dut-1 (RNAi)-induced abnormal vulval organogenesis protruding vulva (Pvl), whereas the apn-1 mutants did not. The increase in Pvl was dependent on UNG-1 and CHK-2. Methyl viologen, ndx-1 (RNAi) and ndx-2 (RNAi) enhanced the incidence of Pvl among exo-3 mutants only when combined with dut-1 (RNAi). This further increase in Pvl incidence was independent of NTH-1. These results indicate that EXO-3 prevents developmental delay and Pvl in C. elegans, which are induced via DNA glycosylase-initiated checkpoint activation.

ATM Induces Cell Death with Autophagy in Response to H2O2 Specifically in Caenorhabditis elegans Nondividing Cells.

INTRODUCTION: Ataxia-telangiectasia-mutated (ATM) kinase is a master regulator of the DNA damage response and is directly activated by reactive oxygen species (ROSs) in addition to DNA double-stranded breaks. However, the physiological function of the response to ROSs is not understood. PURPOSE: In the present study, we investigated how ATM responds to ROSs in Caenorhabditis elegans (C. elegans). MATERIALS AND METHODS: First, we measured sensitivities of larvae to DNA-damaging agents and ROSs. Next, we analyzed the drug sensitivities of fully matured adult worms, which consist of nondividing somatic cells. Dead cell staining with acridine orange was performed to visualize the dead cells. In addition, we performed GFP reporter assays of lgg-1, an autophagy-related gene, to determine the types of cell death. RESULTS: atm-1(tm5027) larvae showed a wide range of sensitivities to both DNA-damaging agents and ROSs. In contrast, fully matured adult worms, which consist of nondividing somatic cells, showed sensitivity to DNA-damaging agent, NaHSO3, but they showed resistance to H2O2. Dead cell staining and GFP reporter assays of lgg-1 suggest that C. elegans ATM-1 induces the cell death with autophagy in intestinal cells in response to H2O2. CONCLUSION: We revealed that ATM induces cell death in response to H2O2.

CiAPEX2 and CiP0, candidates of AP endonucleases in Ciona intestinalis, have 3'-5' exonuclease activity and contribute to protection against oxidative stress.

Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions. AP sites inhibit transcription and DNA replication, and induce cell death. AP endonucleases are key enzymes in AP site repair. Several types of AP endonucleases have been reported, such as AP endonuclease 2 (APEX2) and ribosomal protein P0 (P0). However, it is not known how the functions and roles differ among AP endonucleases. To clarify the difference of roles among AP endonucleases, we conducted biochemical analysis focused on APEX2 and P0 homologues in Ciona intestinalis. Amino acid sequence analysis suggested that CiAPEX2 and CiP0 are AP endonuclease homologues. Although we could not detect AP endonuclease or 3'-phosphodiesterase activity, these two purified proteins exhibited 3'-5' exonuclease activity. This 3'-5' exonuclease activity was sensitive to ethylenediaminetetraacetic acid (EDTA), and the efficiency of this activity was influenced by the 3'-terminus of substrate DNA. Both CiAPEX2 and CiP0 degraded not only a 5'-protruding DNA end, but also nicked DNA, which is generated through AP endonuclease 1 (APEX1) cleavage. These two genes partially complemented the growth rate of AP endonuclease-deficient Escherichia coli treated with hydrogen peroxide. These results indicate that 3'-5' exonuclease activity is an evolutionarily conserved enzymatic activity of APEX2 and P0 homologues and this enzymatic activity may be important for AP endonucleases.

NBS1 is regulated by two kind of mechanisms: ATM-dependent complex formation with MRE11 and RAD50, and cell cycle-dependent degradation of protein.

Nijmegen breakage syndrome (NBS), a condition similar to Ataxia-Telangiectasia (A-T), is a radiation-hypersensitive genetic disorder showing chromosomal instability, radio-resistant DNA synthesis, immunodeficiency, and predisposition to malignances. The product of the responsible gene, NBS1, forms a complex with MRE11 and RAD50 (MRN complex). The MRN complex is necessary for the DNA damage-induced activation of ATM. However, the regulation of MRN complex formation is still unclear. Here, we investigated the regulatory mechanisms of MRN complex formation. We used an immunoprecipitation assay to determine whether levels of the MRN complex were increased by radiation-induced DNA damage and found that the levels of these proteins and their mRNAs did not increase. ATM-dependent phosphorylation of NBS1 contributed to the DNA damage-induced MRN complex formation. However, pre-treatment of cells with an ATM-specific inhibitor did not affect homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. G0 phase cells, decreasing NBS1 and HR activity but not NHEJ, gained HR-related chromatin association of RAD51 by overexpression of NBS1, suggesting that the amount of NBS1 may be important for repressing accidental activation of HR. These evidences suggest that NBS1 is regulated by two kind of mechanisms: complex formation dependent on ATM, and protein degradation mediated by an unknown MG132-resistant pathway. Such regulation of NBS1 may contribute to cellular responses to double-strand breaks.

FUdR extends the lifespan of the short-lived AP endonuclease mutant in Caenorhabditis elegans in a fertility-dependent manner.

The anticancer drug 5-fluorouracil (5-FU) and its metabolite 5-fluoro-2'-deoxyuridine (FUdR) inhibit thymidylate synthase and induce uracil bases in DNA. FUdR is commonly used for inhibiting fertility when measuring the lifespan of the nematode Caenorhabditis elegans. However, it is not known whether DNA damage induced by FUdR affects lifespan. EXO-3 is an apurinic/apyrimidinic endonuclease in C. elegans, and we reported previously that deletion of the exo-3 gene causes reproductive abnormalities and decreased lifespan. In this study, we found that FUdR extended the lifespan of exo-3 mutants. We measured the lifespan of multiple germline mutants to examine whether this lifespan extension effect was dependent on fertility. In the presence of a fem-1 mutation, which causes a deficiency in sperm production, FUdR did not extend the lifespan of the exo-3 mutant. In glp-1 mutants, which do not develop gonads, the exo-3 mutant was not short-lived, and FUdR did not extend its lifespan. These results suggest that the lifespan extension effect of FUdR depends on fertility and the presence of gonads. fem-3 mutants, which do not produce oocytes, had increased lifespan in the presence of FUdR, independent of the exo-3 mutation. It is possible that the fem-3 mutant was susceptible to the lifespan extension effect of FUdR. From these results, we suggest that FUdR affects the lifespan of C. elegans in two ways: by interfering with fertility, which extends lifespan, and by inducing DNA base damage, which reduces lifespan.

pqiABC and yebST, Putative mce Operons of Escherichia coli, Encode Transport Pathways and Contribute to Membrane Integrity.

The membranes of single-cell organisms are crucial as the first line of defense. The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharides (LPSs) and phospholipids are localized in the outer and inner leaflet, respectively. This asymmetry is important for membrane integrity. In Escherichia coli, the Mla transport pathway maintains this asymmetry by removing phospholipids from the outer leaflet. The MlaD component of this system is a mammalian cell entry (MCE) domain protein, and E. coli has two other MCE domain proteins of unknown function (PqiB and YebT). Here, we show that these two proteins are components of novel transport pathways that contribute to membrane integrity. The pqiAB operon is regulated by SoxS and RpoS. The yebST operon contains pqiAB homologues. Here, we found a third member of the pqi operon, ymbA (pqiC). A PqiB-PqiC complex bridges the inner and the outer membrane, and in other bacteria, pqiBC genes are located in operons together with transporter proteins. We show here that simultaneous deletion of pqiABC and yebST operons in an Δmla background rendered cells more sensitive to SDS-EDTA, and the SDS-EDTA sensitivity of mla mutants was rescued by additional copies of pqiABC We also found that the yebST operon was induced by a defect in LPS molecules. In conclusion, PqiABC and YebST are novel transport pathways related to the Mla transport pathway and important for membrane integrity. IMPORTANCE: Membranes of bacteria are crucial for stress resistance. The composition of the E. coli outer membrane is asymmetric, with asymmetry maintained by the Mla ABC transport pathway. We propose that the stress-inducible pqiABC operon and homologous yebST operon, both of previously unknown function, encode transport pathway proteins related to the Mla transport pathway. Deletion of these operons rendered cells more sensitive to membrane stress, and additional copies of pqiABC suppressed the SDS-EDTA sensitivity of mla mutant strains. We found that yebS'-'lacZ fusion was activated in mutant strains with defective LPS molecules.

A novel DNA damage response mediated by DNA mismatch repair in Caenorhabditis elegans: induction of programmed autophagic cell death in non-dividing cells.

DNA mismatch repair (MMR) contributes to genome integrity by correcting errors of DNA polymerase and inducing cell death in response to DNA damage. Dysfunction of MMR results in increased mutation frequency and cancer risk. Clinical researches revealed that MMR abnormalities induce cancers of non-dividing tissues, such as kidney and liver. However, how MMR suppresses cancer in non-dividing tissues is not understood. To address that mechanism, we analyzed the roles of MMR in non-dividing cells using Caenorhabditis elegans (C. elegans), in which all somatic cells are non-dividing in the adult stage. In this study, we used stable MMR-mutant lines with a balancer chromosome. First, we confirmed that deficiency of MMR leads to resistance to various mutagens in C. elegans dividing cells. Next, we performed drug resistance assays, and found that MMR-deficient adult worms were resistant to SN1-type alkylating and oxidizing agents. In addition, dead cell staining and reporter assays of an autophagy-related gene demonstrated that the cell death was autophagic cell death. Interestingly, this autophagic cell death was not suppressed by caffeine, implying that MMR induces death of non-dividing cells in an atl-1-independent manner. Hence, we propose the hypothesis that MMR prevents cancers in non-dividing tissues by directly inducing cell death.

Caenorhabditis elegans EXO-3 contributes to longevity and reproduction: differential roles in somatic cells and germ cells.

Apurinic/apyrimidinic (AP) sites are the major DNA damage generated continuously even under normal conditions, and inhibit DNA replication/transcription. AP endonucleases are ubiquitous enzymes required for the repair of AP sites and 3' blocking ends, but their physiological roles in multicellular organisms are not fully understood. In this study, we investigated how an AP endonuclease functions in a multicellular organism (Caenorhabditis elegans (C. elegans)). EXO-3 is one of the AP endonucleases in C. elegans. Using an exo-3 mutant worm, we found that deletion of the exo-3 gene caused shortened lifespan in an ung-1-dependent manner. UNG-1 is a uracil DNA glycosylase in C. elegans, and the present finding suggested that UNG-1 is the major producer of AP sites that affects lifespan, and EXO-3 contributes to longevity by completing the repair of uracil. Next we found that the exo-3 gene was abundantly expressed in the gonads, and AP sites in the gonad were efficiently repaired, suggesting that EXO-3 functioned particularly in the gonad. Deletion of the exo-3 gene resulted in a significant decrease in self-brood size. This was rescued by deficiency of NTH-1, which is a bifunctional DNA glycosylase in C. elegans that recognizes oxidative base damage. This result suggested that the major substrate of EXO-3 in the gonad was 3' blocking end generated by NTH-1, and that EXO-3 played an important role in reproduction. A contribution of EXO-3 to reproduction was also suggested by our finding here that the decrease of self-brood size of the exo-3 mutant became more marked when worms were treated with methyl methanesulfonate (MMS) and sodium bisulfite (NaHSO3). This study demonstrated differential roles of EXO-3 in somatic cells and germ cells.

An increase of oxidised nucleotides activates DNA damage checkpoint pathway that regulates post-embryonic development in Caenorhabditis elegans.

8-Oxo-dGTP, an oxidised form of dGTP generated in the nucleotide pool, can be incorporated opposite adenine or cytosine in template DNA, which can in turn induce mutations. In this study, we identified a novel MutT homolog (NDX-2) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. In addition, we found that NDX-1, NDX-2 and NDX-4 proteins have 8-oxo-GTPase or 8-oxo-GDPase activity. The sensitivity of ndx-2 knockdown C. elegans worms to methyl viologen and menadione bisulphite was increased compared with that of control worms. This sensitivity was rescued by depletion of chk-2 and clk-2, suggesting that growth of the worms is regulated by the checkpoint pathway in response to the accumulation of oxidised nucleotides. Moreover, we found that the sensitivity to menadione bisulphite of ndx-1 and ndx-2-double knockdown worms was enhanced by elimination of XPA-1, a factor involved in nucleotide excision repair. The rescue effect by depletion of chk-2 and clk-2 was limited in the xpa-1 mutant, suggesting that the chk-2 and clk-2 checkpoint pathway is partially linked to the function of XPA-1.

Extension of lifespan and protection against oxidative stress by an antioxidant herb mixture complex (KPG-7) in Caenorhabditis elegans.

Excessive generation of reactive oxygen species within cells results in oxidative stress. Furthermore, accumulation of reactive oxygen species has been shown to reduce cell longevity. Many dietary supplements are believed to have anti-aging effects. The herb mixture KPG-7 contains several components with antioxidant activity. We aim to clarify the mechanisms responsible for the antioxidant activity of KPG-7 and to establish whether KPG-7 has an anti-aging effect. We examined whether dietary supplementation with KPG-7 could provide protection against oxidative stress, extend lifespan, and delay aging in Caenorhabditis elegans (C. elegans). We found that KPG-7 extended lifespan and delayed aging in adult C. elegans. The expression of oxidation resistance 1 protein was induced by juglone and this effect was significantly suppressed in KPG-7-treated. In addition, the amount of oxidized protein was significantly lower in KPG-7-treated worms than untreated worms. Furthermore, locomotive activity was increased in C. elegans at 3 days of age following the treatment with KPG-7. On the other hand, the level of cellular ATP was lower at 3 days of age in worms treated with KPG-7 than in untreated worms. KPG-7 increases lifespan and delays aging in C. elegans, well corresponding to its activity to protect against oxidative stress.

Escherichia coli pyruvate:flavodoxin oxidoreductase, YdbK - regulation of expression and biological roles in protection against oxidative stress.

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.

Structural and functional properties of CiNTH, an endonuclease III homologue of the ascidian Ciona intestinalis: critical role of N-terminal region.

Oxidatively damaged bases in DNA can cause cell death, mutation and/or cancer induction. To overcome such deleterious effects of DNA base oxidation, cells are equipped with base excision repair (BER) initiated by DNA glycosylases. Endonuclease III (Nth), a major DNA glycosylase, mainly excises oxidatively damaged pyrimidines from DNA. The aims of this study were to obtain an overview of the repair mechanism of oxidatively damaged bases and to elucidate the function of BER in maintaining genome stability during embryogenesis and development. In this study, we used the ascidian Ciona intestinalis because at every developmental stage it is possible to observe the phenotype of individuals with DNA damage or mutations. Sequence alignment analysis revealed that the amino acid sequence of Ciona intestinalis Nth homologue (CiNTH) had high homology with those of Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and human Nth homologues. It was evident that two domains, the Helix-hairpin-Helix and 4Fe-4S cluster domains that are critical regions for the Nth activity, are well conserved in CiNTH. CiNTH efficiently complemented the sensitivity of E. coli nth nei mutant to H(2)O(2). CiNTH was bifunctional, with DNA glycosylase and AP lyase activities. It removed thymine glycol, 5-formyluracil and 8-oxoguanine paired with G from DNA via a ß-elimination reaction. Interestingly, the N-terminal 44 amino acids were essential for the DNA glycosylase activity of CiNTH.

Mitochondria-targeted superoxide dismutase (SOD2) regulates radiation resistance and radiation stress response in HeLa cells.

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. MitoSox(TM) 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.