Shigenori Iwai

Osaka University, Suika, Ōsaka, Japan

Are you Shigenori Iwai?

Claim your profile

Publications (173)1041.24 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apo-protein, and substrate, so that only the initial, ultrafast, photon-chromophore reaction, and the final, slow, protein conformational change provide separate, non-overlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening timescales still cannot be fully deconvoluted. We overcome the problem by using novel method to selectively isotope-label the apo-protein but not the FAD cofactor. This enabled the FTIR signals to be separated from apo-protein, FAD cofactor, and DNA substrate. Consequently, comprehensive structure-functional study by Fourier transform infrared (FTIR) spectroscopy of the E. coli CPD-Photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair which revealed protein dynamics for both processes, beyond simple one-electron reduction and ejection, respectively. The FTIR data suggests that the synergistic cofactor-protein partnership in CPD-PHR linked to changes in FAD shape upon one-electron reduction may be coordinated with conformational changes in the apo-protein, allowing it to fit the DNA substrate. Activation of CPD-PHR chromophore primes the apo-protein for subsequent DNA repair, suggesting that CPD-PHR is not simply an electron-ejecting structure. When FAD is activated, changes in its structure may trigger coordinated conformational changes in the apo-protein and thymine carbonyl of the substrate, highlighting the role of Glu275. In contrast, during DNA repair and release processes, primary conformational changes occur in the enzyme and DNA substrate, with little contribution from the FAD cofactor and surrounding amino acid residues.
    Biochemistry 08/2014; · 3.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.
    PLoS ONE 01/2014; 9(1):e85896. · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, G·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions.
    Scientific reports. 01/2014; 4:5220.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6-4) photoproduct and a fluorophore-quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3 h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6-4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells.
    Scientific reports. 01/2014; 4:5578.
  • Yukiko Kametani, Shigenori Iwai, Isao Kuraoka
    [Show abstract] [Hide abstract]
    ABSTRACT: Biological risk assessment studies of chemical substances that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication, but also with transcription. There is no simple method for the detection of the DNA lesion-induced inhibition of transcription. Here, we report an assay for estimating the toxicity of chemical substances by visualizing transcription in mammalian cells using nucleotide analog 5-ethynyluridine (EU) and its click chemistry reaction. Ultraviolet light and representative chemical substances (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) of DNA- damaging agents show toxicity, as indicated by RNA synthesis inhibition in response to DNA damage in HeLa cells. Using titanium dioxide, we observed RNA synthesis inhibition in response to the rutile form, but not the anatase form, indicating that rutile titanium dioxide is a toxic substance. Because this method is based on the transcriptional response to DNA lesions, we can use terminally differentiated neuron-like PC12 cells, the differentiation of which can be induced by nerve growth factors, for evaluating chemical substances. Ultraviolet light and some chemicals (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) inhibited RNA synthesis in non-differentiated PC12 cells. Conversely, camptothecin and cisplatin did not inhibit RNA synthesis in differentiated PC12 cells, but 4-nitroquinoline-1-oxide, mitomycin C, and etoposide did. And using titanium dioxide, we did not observed any RNA synthesis inhibition. These data suggest that this method might be used to estimate the potential risk of chemical substances in differentiated mammalian cells, which are the most common cell type found in the human body.
    The Journal of Toxicological Sciences 01/2014; 39(2):293-9. · 1.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2'-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.
    Nucleic Acids Research 10/2013; · 8.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Deamination of DNA bases can create missense mutations predisposing humans to cancer and also interfere with other basic molecular genetic processes; this deamination generates deoxyinosine from deoxyadenosine. In Escherichia coli, the highly conserved endonuclease V is involved in alternative excision repair that removes deoxyinosine from DNA. However, its exact activities and roles in humans are unknown. Here we characterize the FLJ35220 protein, the human homologue of E. coli endonuclease V, hEndoV as a ribonuclease specific for inosine-containing RNA. hEndoV preferentially binds to RNA and efficiently hydrolyses the second phosphodiester bond located 3' to the inosine in unpaired inosine-containing ssRNA regions in dsRNA. It localizes to the cytoplasm in cells. The ribonuclease activity is promoted by Tudor staphylococcal nuclease and detected on inosine-containing dsRNA created by the action of adenosine deaminases acting on RNA. These results demonstrate that hEndoV controls the fate of inosine-containing RNA in humans.
    Nature Communications 08/2013; 4:2273. · 10.74 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: It takes two (photons) to tango: Single-turnover flash experiments showed that the flavoenzyme (6-4) photolyase uses a successive two-photon mechanism to repair the UV-induced T(6-4)T lesion in DNA. The intermediate (X) formed by the first photoreaction is likely to be the oxetane-bridged dimer T(ox)T. The enzyme could stabilize the normally short-lived T(ox)T, allowing repair to be completed by the second photoreaction.
    Angewandte Chemie International Edition 06/2013; · 11.34 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We previously developed a molecular beacon-type probe to detect the strand scission in cellular base excision repair, and found that the phosphodiester linkages in the fluorophore/quencher linkers were cleaved. This reaction was applied to a transfection reporter, which contained the unmodified phosphodiester in the linker to another type of fluorophore. After co-transfection of cells with the probe and the reporter, the signals were used to detect the incision and to confirm the proper transfection, respectively. This method will contribute to the prevention of false-negative results in experiments using molecular beacon-type probes.
    Analytical Biochemistry 05/2013; · 2.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The (6-4) photoproduct is one of the major UV-induced lesions in DNA. We previously showed that hydrolytic ring opening of the 5' base and subsequent hydrolysis of the glycosidic bond of the 3' component occurred when this photoproduct was treated with aqueous NaOH. In this study, we found that another product was obtained when the (6-4) photoproduct was heated at 90 °C for 6 h, in a 0.1 M solution of N,N'-dimethyl-1,2-ethanediamine adjusted to pH 7.4 with acetic acid. An analysis of the chemical structure of this product revealed that the 5' base was intact, whereas the glycosidic bond at the 3' component was hydrolyzed in the same manner. The strand break was detected for a 30-mer oligonucleotide containing the (6-4) photoproduct upon treatment with the above solution or other pH 7.4 solutions containing biogenic amines, such as spermidine and spermine. In the case of spermidine, the rate constant was calculated to be 1.4 × 10(-8) s(-1) at 37 °C. The strand break occurred even when the oligonucleotide was heated at 90 °C in 0.1 M sodium phosphate (pH 7.0), although this treatment produced several types of 5' fragments. The Dewar valence isomer was inert to this reaction. The product obtained from the (6-4) photoproduct-containing 30-mer was used to investigate the enzymatic processing of the 3' end bearing the damaged base and a phosphate. The ERCC1-XPF complex removed several nucleotides containing the damaged base, in the presence of replication protein A.
    Organic & Biomolecular Chemistry 04/2013; · 3.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Photolyases (PHRs) utilize near-ultraviolet (UV)–blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer PHR (CPD-PHR) from Escherichia coli binds flavin adenine dinucleotide (FAD) as a cofactor and 5,10-methenyltetrahydrofolate as a light-harvesting pigment and specifically repairs CPD lesions. By comparison, a second photolyase known as (6–4) PHR, present in a range of higher organisms, uniquely repairs (6–4) PPs. To understand the repair mechanism and the substrate specificity that distinguish CPD-PHR from (6–4) PHR, we applied Fourier transform infrared (FTIR) spectroscopy to bacterial CPD-PHR in the presence or absence of a well-defined DNA substrate, as we have studied previously for vertebrate (6–4) PHR. PHRs show light-induced reduction of FAD, and photorepair by CPD-PHR involves the transfer of an electron from the photoexcited reduced FAD to the damaged DNA for cleaving the dimers to maintain the DNA’s integrity. Here, we measured and analyzed difference FTIR spectra for the photoactivation and DNA photorepair processes of CPD-PHR. We identified light-dependent signals only in the presence of substrate. The signals, presumably arising from a protonated carboxylic acid or the DNA substrate, implicate conformational rearrangements of the protein and substrate during the repair process. Deuterium exchange FTIR measurements of CPD-PHR highlight potential differences in the photoactivation and photorepair mechanisms in comparison to those of (6–4) PHR. Although CPD-PHR and (6–4) PHR appear to exhibit similar overall structures, our studies indicate that distinct conformational rearrangements, especially in the α-helices, are initiated within these enzymes upon binding of their respective DNA substrates.
    Biochemistry 01/2013; 52(6):1019–1027. · 3.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Hydroxyl radicals generate a broad range of DNA lesions in living cells. Cyclopurine deoxynucleosides (cPu) are a biologically significant class of oxidative DNA lesions due to their helical distortion and chemically stability. The cPu on DNA are substrates for the nucleotide excision repair (NER), but not for base excision repair or direct damage reversal. Moreover, these lesions block DNA and RNA polymerases, resulting in cell death. Here we describe the chemical synthesis of 5'S and 5'R isomers of 5',8-cyclodeoxyadenosine triphosphate (cdATP) and demonstrate their ability to be incorporated into DNA by replicative DNA polymerases. DNA synthesis assays revealed that the incorporation of the stereoisomeric cdATPs strongly inhibits DNA polymerase reactions. Surprisingly, the 2 stereoisomers had different mutagenic profiles, since the S isomer of cdATP could be inserted opposite to the dTMP, but the R isomer of cdATP could be inserted opposite to the dCMP. Kinetic analysis revealed that the S isomer of cdATP could be incorporated more efficiently (25.6 µM-1 min-1) than the R isomer (1.13 µM-1 min-1) during DNA synthesis. Previous data showed that the S isomer in DNA blocked DNA synthesis and the exonuclease activity of DNA polymerase, and is less efficiently repaired by NER. This indicates that the S isomer has a tendency to accumulate on the genome DNA, and as such, the S isomer of cdATP may be a candidate cytotoxic drug.
    Chemical Research in Toxicology 11/2012; · 3.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Oxidative DNA lesions inhibit the transcription of RNA polymerase II, but in the presence of transcription elongation factors, the transcription can bypass the lesions. Single-subunit mitochondrial RNA polymerase (mtRNAP) catalyses the synthesis of essential transcripts in mitochondria where reactive oxidative species (ROS) are generated as by-products. The occurrence of RNA synthesis by mtRNAP at oxidative DNA lesions remains unknown. Purified mtRNAP and a complex of RNA primer/DNA template containing a single DNA lesion, such as ROS-induced 8-oxoguanine (8-oxoG), two isomeric thymine glycols (5R-Tg or 5S-Tg), the UV-induced cis-syn cyclobutane pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidone photoproduct (6-4pp), or a spontaneous common DNA lesion, a base-loss-induced apurinic/apyrimidinic (AP) site, were used for in vitro RNA synthesis assays. In this report, we show that mtRNAP bypassed the oxidative DNA lesions of non-bulky 8-oxoG and 5R-Tg and 5S-Tg with pausing sites but did not bypass the UV-induced DNA lesions and the AP site. The bacteriophage T7 phage RNA polymerase, which is homologous to mtRNAP, bypassed 8-oxoG but stalled at 5R-Tg and 5S-Tg. As expected, although translesion RNA synthesis in 8-oxoG on the DNA templates generated incorrect transcripts with a G:C to T:A transversion, the synthesis in Tg could lead to the correct transcripts with no transcriptional mutagenesis. Collectively, these data suggest that mtRNAP may tolerate the mitochondrial genome containing oxidative DNA lesions induced by ROS from the side effects of an ATP generation reaction.
    Mutagenesis 10/2012; · 3.50 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.
    Organic & Biomolecular Chemistry 03/2012; 10(11):2318-25. · 3.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.
    Genes to Cells 03/2012; 17(3):173-85. · 2.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.
    PLoS ONE 01/2012; 7(12):e52472. · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ozone depletion increases terrestrial solar ultraviolet B (UV-B; 280-315 nm) radiation, intensifying the risks plants face from DNA damage, especially covalent cyclobutane pyrimidine dimers (CPD). Without efficient repair, UV-B destroys genetic integrity, but plant breeding creates rice cultivars with more robust photolyase (PHR) DNA repair activity as an environmental adaptation. So improved strains of Oryza sativa (rice), the staple food for Asia, have expanded rice cultivation worldwide. Efficient light-driven PHR enzymes restore normal pyrimidines to UV-damaged DNA by using blue light via flavin adenine dinucleotide to break pyrimidine dimers. Eukaryotes duplicated the photolyase gene, producing PHRs that gained functions and adopted activities that are distinct from those of prokaryotic PHRs yet are incompletely understood. Many multicellular organisms have two types of PHR: (6-4) PHR, which structurally resembles bacterial CPD PHRs but recognizes different substrates, and Class II CPD PHR, which is remarkably dissimilar in sequence from bacterial PHRs despite their common substrate. To understand the enigmatic DNA repair mechanisms of PHRs in eukaryotic cells, we determined the first crystal structure of a eukaryotic Class II CPD PHR from the rice cultivar Sasanishiki. Our 1.7 Å resolution PHR structure reveals structure-activity relationships in Class II PHRs and tuning for enhanced UV tolerance in plants. Structural comparisons with prokaryotic Class I CPD PHRs identified differences in the binding site for UV-damaged DNA substrate. Convergent evolution of both flavin hydrogen bonding and a Trp electron transfer pathway establish these as critical functional features for PHRs. These results provide a paradigm for light-dependent DNA repair in higher organisms.
    Journal of Biological Chemistry 12/2011; 287(15):12060-9. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.
    Cell 11/2011; 147(5):1024-39. · 31.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Photolyases (PHRs) are DNA repair proteins that revert UV-induced photoproducts, either cyclobutane pyrimidine dimers (CPD) or (6-4) photoproducts (PPs), into normal bases to maintain genetic integrity. The (6-4) PHR must catalyze not only covalent bond cleavage but also hydroxyl or amino group transfer, yielding a more complex mechanism than that postulated for CPD PHR. Building upon recently established light-induced difference FTIR spectroscopy of Xenopus (6-4) PHR, we now utilize 15N3′-labeled (6-4) PP to identify vibrational modes of (6-4) PP upon repair. We successfully assign two and three vibrational bands for the (6-4) PP and the repaired thymine, respectively. Thus, the present FTIR spectroscopy is sensitive enough to distinguish a single nitrogen atom (15N versus 14N) among >6700 atoms in the enzyme–substrate complex.
    Journal of Physical Chemistry Letters 10/2011; 2(21):2774–2777. · 6.59 Impact Factor
  • Yoko Morita, Shigenori Iwai, Isao Kuraoka
    [Show abstract] [Hide abstract]
    ABSTRACT: To date, biological risk assessment studies of chemicals that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication but also with transcription. Therefore, detecting the damaging effects of DNA lesions during transcription might be important for estimating the safety of chemical mutagens and carcinogens. However, methods to address these effects have not been developed. Here, we report a simple, non-isotopic method for determining the toxicity of chemical agents by visualizing transcription in a mammalian cell system. The method is based on the measurement of the incorporation of bromouridine (as the uridine analogue) into the nascent RNA during RNA synthesis inhibition (RSI) induced by the stalling of RNA polymerases at DNA lesions on the transcribed DNA strand, which triggers transcription-coupled nucleotide excision repair (TC-NER). When we tested chemical agents (camptothecin, etoposide, 4-nitroquinoline-1-oxide, mitomycin C, methyl methanesulfonate, and cisplatin) in HeLa cells by the method, RSI indicative of genomic toxicity was observed in the nucleoli of the tested cells. This procedure provides the following advantages: 1) it uses common, affordable mammalian cells (HeLa cells, WI38VA13 cells, human dermal fibroblasts, or Chinese hamster ovary cells) rather than genetically modified microorganisms; 2) it can be completed within approximately 8 hr after the cells are prepared because RNA polymerase responses during TC-NER are faster than other DNA damage responses (replication, recombination, and apoptosis); and 3) it is safe because it uses non-radioactive bromouridine and antibodies to detect RNA synthesis on undamaged transcribed DNA strands.
    The Journal of Toxicological Sciences 10/2011; 36(5):515-21. · 1.38 Impact Factor

Publication Stats

7k Citations
1,041.24 Total Impact Points

Institutions

  • 1985–2014
    • Osaka University
      • • Department of Chemistry
      • • Division of Gene Therapy Science
      • • Division of Cellular and Molecular Biology
      • • Division of Molecular Pharmaceutical Science
      Suika, Ōsaka, Japan
  • 2013
    • Gakushuin University
      Edo, Tōkyō, Japan
  • 2011
    • Nagoya Institute of Technology
      • Department of Frontier Materials
      Nagoya-shi, Aichi-ken, Japan
  • 2009–2011
    • Friedrich Miescher Institute for Biomedical Research
      Bâle, Basel-City, Switzerland
    • National Hospital Organization Kyushu Cancer Center
      Hukuoka, Fukuoka, Japan
    • Kobe University
      • Biosignal Research Center
      Kōbe, Hyōgo, Japan
  • 2003–2008
    • Hiroshima University
      • • Division of Mathematical and Life Sciences
      • • Graduate School of Science
      Hiroshima-shi, Hiroshima-ken, Japan
  • 2006
    • University of Pittsburgh
      • Hillman Cancer Center
      Pittsburgh, PA, United States
  • 2000–2006
    • National Institute of Child Health and Human Development
      Maryland, United States
  • 2001–2005
    • RIKEN
      • Laboratory For Physical Biology
      Wako, Saitama-ken, Japan
  • 2002–2003
    • The University of Tokyo
      • Department of Chemistry and Biotechnology
      Edo, Tōkyō, Japan
  • 1998–2001
    • Kyoto University
      • Radiation Biology Center
      Kyoto, Kyoto-fu, Japan
  • 1987–1997
    • Hokkaido University
      • Faculty of Pharmaceutical Sciences
      Sapporo-shi, Hokkaido, Japan
  • 1994–1995
    • Tokyo University of Science
      • Department of Pharmaceutical Sciences
      Tokyo, Tokyo-to, Japan