Shigenori Iwai

Osaka University, Suika, Ōsaka, Japan

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Publications (206)1236.61 Total impact

  • Yuina Sonohara · Shigenori Iwai · Isao Kuraoka ·
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    ABSTRACT: Introduction: A wide variety of DNA lesions such as ultraviolet light-induced photoproducts and chemically induced bulky adducts and crosslinks (intrastrand and interstrand) interfere with replication and lead to mutations and cell death. In the human body, these damages may cause cancer, inborn diseases, and aging. So far, mutation-related actions of DNA polymerases during replication have been intensively studied. However, DNA lesions also block RNA synthesis, making the detection of their effects on transcription equally important for chemical safety assessment. Previously, we established an in vivo method for detecting DNA damage induced by ultraviolet light and/or chemicals via inhibition of RNA polymerase by visualizing transcription. Results: Here, we present an in vitro method for detecting the effects of chemically induced DNA lesions using in vitro transcription with T7 RNA polymerase and real-time reverse transcription polymerase chain reaction (PCR) based on inhibition of in vitro RNA synthesis. Conventional PCR and real-time reverse transcription PCR without in vitro transcription can detect DNA lesions such as complicated cisplatin DNA adducts but not UV-induced lesions. We found that only this combination of in vitro transcription and real-time reverse transcription PCR can detect both cisplatin- And UV-induced DNA lesions that interfere with transcription. Conclusions: We anticipate that this method will be useful for estimating the potential transcriptional toxicity of chemicals in terminally differentiated cells engaged in active transcription and translation but not in replication.
    Genes and Environment 12/2015; 37(1). DOI:10.1186/s41021-015-0014-8
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    ABSTRACT: Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and Xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates, but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.
    Carcinogenesis 11/2015; DOI:10.1093/carcin/bgv159 · 5.33 Impact Factor
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    ABSTRACT: UV-DDB, an initiation factor for the nucleotide excision repair pathway, recognizes 6-4PP lesions through a base flipping mechanism. As genomic DNA is almost entirely accommodated within nucleosomes, the flipping of the 6-4PP bases is supposed to be extremely difficult if the lesion occurs in a nucleosome, especially on the strand directly contacting the histone surface. Here we report that UV-DDB binds efficiently to nucleosomal 6-4PPs that are rotationally positioned on the solvent accessible or occluded surface. We determined the crystal structures of nucleosomes containing 6-4PPs in these rotational positions, and found that the 6-4PP DNA regions were flexibly disordered, especially in the strand exposed to the solvent. This characteristic of 6-4PP may facilitate UV-DDB binding to the damaged nucleosome. We present the first atomic-resolution pictures of the detrimental DNA cross-links of neighboring pyrimidine bases within the nucleosome, and provide the mechanistic framework for lesion recognition by UV-DDB in chromatin.
    Scientific Reports 11/2015; 5:16330. DOI:10.1038/srep16330 · 5.58 Impact Factor
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    ABSTRACT: Bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM) bisintercalates to DNA and is a potential anti-cancer therapeutic. In an attempt to elucidate the mechanism(s) underlying the potential of BNIPDaCHM; earlier work was extended to investigate its effect on DNA damage and repair as well as cell cycle modulation, in a triple negative breast cancer (TNBC) cell line in vitro. BNIPDaCHM significantly decreased cell viability in a concentration (≥5 μM) and time (≥24 hours) dependent manner. The mechanism of this growth inhibition involved alterations to cell cycle progression, an increase in the sub-G1 population and changes to plasma membrane integrity/permeability observed by flow cytometry and fluorescence microscopy with acridine orange/ethidium bromide staining. Using single cell gel electrophoresis (Comet assay) and fluorescence microscopy to detect γ-H2AX-foci expression; it was found that after 4 hours, ≥ 0.1 μM BNIPDaCHM treatment-induced significant DNA double strand breaks (DSBs). Moreover, exposure to a non-genotoxic concentration of BNIPDaCHM induced a significant decrease in the repair of oxidative DNA strand breaks induced by hydrogen peroxide. Also, BNIPDaCHM-treatment induced a significant time dependent increase in p21(Waf/Cip1) mRNA expression but, did not alter p53 mRNA expression. In conclusion, BNIPDaCHM treatment in MDA-MB-231 cells was associated with a significant induction of DNA DSBs and inhibition of DNA repair at non-genotoxic concentrations via p53-independent expression of p21(Waf1/Cip1). The latter may be a consequence of novel interactions between BNIPDaCHM and MDA-MB-231 cells which adds to the spectrum of therapeutically relevant activities that may be exploited in the future design and development of naphthalimide-based therapeutics.
    Chemico-biological interactions 10/2015; 242. DOI:10.1016/j.cbi.2015.10.017 · 2.58 Impact Factor
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    Pavel Müller · Junpei Yamamoto · Ryan Martin · Shigenori Iwai · Klaus Brettel ·
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    ABSTRACT: A 4th electron transferring tryptophan in animal cryptochromes and (6-4) photolyases is discovered and functionally analyzed by transient absorption. It yields a much longer-lived flavin-tryptophan radical pair than the mere tryptophan triad in related flavoproteins, questioning the putative role of the primary light reaction of cryptochrome in animal magnetoreception.
    Chemical Communications 08/2015; 51:15502-15505. DOI:10.1039/C5CC06276D · 6.83 Impact Factor
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    ABSTRACT: Imidazole was tethered to the C5 position of thymine in the ATP-binding DNA aptamer with two types of linkers, and the affinities of each aptamer for ATP and AMP were determined by surface plasmon resonance measurements. The imidazole-tethered aptamers exhibited higher affinity for ATP, almost independently of the linker structure or the modification site.
    The Analyst 07/2015; 140(17). DOI:10.1039/C5AN01347J · 4.11 Impact Factor
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    ABSTRACT: The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER.
    Scientific Reports 06/2015; 5:10984. DOI:10.1038/srep10984 · 5.58 Impact Factor
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    ABSTRACT: Topoisomerase 1 (Top1) is the intercellular target of camptothecins (CPTs). CPT blocks DNA religation in the Top1-DNA complex and induces Top1-attached nick DNA lesions. In this study, we demonstrate that excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease and replication protein A (RPA) participate in the repair of Top1-attached nick DNA lesions together with other nucleotide excision repair (NER) factors. ERCC1-XPF shows nuclease activity in the presence of RPA on a 3'-phosphotyrosyl bond nick-containing DNA (Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. In addition, ERCC1-XPF and RPA form a DNA/protein complex on the nick DNA substrate in vitro, and co-localize in CPT-treated cells in vivo. Moreover, the DNA repair synthesis of Tyr-nick DNA lesions occurred in the presence of NER factors, including ERCC1-XPF, RPA, DNA polymerase delta, flap endonuclease 1 and DNA ligase 1. Therefore, some of the NER repair machinery might be an alternative repair pathway for Top1-attached nick DNA lesions. Clinically, these data provide insights into the potential of ERCC1 as a biomarker during CPT regimens. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email:
    Carcinogenesis 05/2015; 36(8). DOI:10.1093/carcin/bgv078 · 5.33 Impact Factor
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    ABSTRACT: In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects. © 2015 Tsaalbi-Shtylik et al.
    The Journal of Cell Biology 04/2015; 209(1):33-46. DOI:10.1083/jcb.201408017 · 9.83 Impact Factor
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    ABSTRACT: Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6-4) photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6-4) photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.
    PLoS ONE 02/2015; 10(2):e0117798. DOI:10.1371/journal.pone.0117798 · 3.23 Impact Factor
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    ABSTRACT: In mammalian nucleotide excision repair, the DDB1-DDB2 complex recognizes UV-induced DNA photolesions and facilitates recruitment of the XPC complex. Upon binding to damaged DNA, the Cullin 4 ubiquitin ligase associated with DDB1-DDB2 is activated and ubiquitinates DDB2 and XPC. The structurally disordered N-terminal tail of DDB2 contains seven lysines identified as major sites for ubiquitination that target the protein for proteasomal degradation; however, the precise biological functions of these modifications remained unknown. By exogenous expression of mutant DDB2 proteins in normal human fibroblasts, here we show that the N-terminal tail of DDB2 is involved in regulation of cellular responses to UV. By striking contrast with behaviors of exogenous DDB2, the endogenous DDB2 protein was stabilized even after UV irradiation as a function of the XPC expression level. Furthermore, XPC competitively suppressed ubiquitination of DDB2 in vitro, and this effect was significantly promoted by centrin-2, which augments the DNA damage-recognition activity of XPC. Based on these findings, we propose that in cells exposed to UV, DDB2 is protected by XPC from ubiquitination and degradation in a stochastic manner; thus XPC allows DDB2 to initiate multiple rounds of repair events, thereby contributing to the persistence of cellular DNA repair capacity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 01/2015; 43(3). DOI:10.1093/nar/gkv038 · 9.11 Impact Factor
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    ABSTRACT: Photolyases (PHRs) utilize near UV/blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer (CPD)-PHR binds flavin adenine dinucleotide (FAD) as a cofactor and repairs CPD lesions in double-stranded DNA. To understand the activation and repair mechanism of CPD-PHR, we applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to CPD-PHR, whose signals were identified by use of isotope-labeling. To further investigate the enzymatic function, here we study the activation and repair mechanism of CPD-PHR with the substrate in single strand DNA, and the obtained FTIR spectra are compared with those in double-stranded DNA, the natural substrate. The difference spectra of photoactivation, the fully-reduced (FADH-) minus semiquinone (FADH•) spectra, are almost identical in the presence of single strand and double-stranded DNA, except for slight spectral modification in the amide-I region. On the other hand, the difference spectra of photorepair were highly substrate dependent. Strong bands of the C=O stretch (1,720-1,690 cm-1) and phosphate vibrations (1,090-1,060 cm-1) of double-stranded DNA may have disappeared in the case of single strand DNA. However, an isotope-labeled enzyme study revealed that spectral features upon DNA repair are similar between both substrates, and the main reason for the apparent spectral difference originates from structural flexibility of DNA after repair.
    BIOPHYSICS 01/2015; 11:39-45. DOI:10.2142/biophysics.11.39
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    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; 53(37). DOI:10.1021/bi500638b · 3.02 Impact Factor
  • J Chiba · S Aoki · J Yamamoto · S Iwai · M Inouye ·
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    ABSTRACT: We report bending flexibility of damaged duplexes possessing an apurinic/apyrimidinic (AP) site analogue, a cyclobutane pyrimidine dimer (CPD), and a pyrimidine(6-4)pyrimidone photoproduct (6-4PP). Based on the electrochemical evaluation on electrodes, the duplex flexibilities of the lesions increased in the following order: CPD < AP < 6-4PP. We discussed the possibility that the emerging local flexibility might be a good sign for UV-damaged DNA-binding proteins on duplexes.
    Chemical Communications 08/2014; 50(76). DOI:10.1039/c4cc04513k · 6.83 Impact Factor
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    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 07/2014; 4:5578. DOI:10.1038/srep05578 · 5.58 Impact Factor
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    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 06/2014; 4:5220. DOI:10.1038/srep05220 · 5.58 Impact Factor
  • Yukiko Kametani · Shigenori Iwai · Isao Kuraoka ·
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    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 03/2014; 39(2):293-9. DOI:10.2131/jts.39.293 · 1.29 Impact Factor
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    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. DOI:10.1371/journal.pone.0085896 · 3.23 Impact Factor
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    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; 42(3). DOI:10.1093/nar/gkt1039 · 9.11 Impact Factor

Publication Stats

11k Citations
1,236.61 Total Impact Points


  • 1985-2015
    • Osaka University
      • • Graduate School of Engineering Sciences
      • • Department of Chemistry
      • • Division of Molecular Pharmaceutical Science
      Suika, Ōsaka, Japan
  • 2010-2013
    • Gakushuin University
      Edo, Tōkyō, Japan
  • 2004-2010
    • Hiroshima University
      • • Division of Mathematical and Life Sciences
      • • Graduate School of Science
      Hirosima, Hiroshima, Japan
  • 2009
    • National Hospital Organization Kyushu Cancer Center
      Hukuoka, Fukuoka, Japan
  • 2007
    • National Research Council
      Bari, Apulia, Italy
  • 1986-2004
    • The University of Tokyo
      • • Department of Chemistry and Biotechnology
      • • Department of Medical Engineering
      • • Institute of Medical Science
      白山, Tōkyō, Japan
  • 2002
    • Marine Biological Research Institute of Japan Co., Ltd.
      Edo, Tōkyō, Japan
  • 2000
    • Hyogo College of Medicine
      Nishinomiya, Hyōgo, Japan
  • 1997-1999
    • Japan Research Institute
      Ōsaka, Ōsaka, Japan
  • 1998
    • Kyoto University
      • Radiation Biology Center
      Kioto, Kyōto, Japan
  • 1987-1997
    • Hokkaido University
      • Faculty of Pharmaceutical Sciences
      Sapporo, Hokkaido, Japan