22 results on '"Seiichiro Kizaki"'
Search Results
2. Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures
- Author
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Yang Jiang, Cécile Mingard, Sabrina M. Huber, Vakil Takhaveev, Maureen McKeague, Seiichiro Kizaki, Mirjam Schneider, Nathalie Ziegler, Vera Hürlimann, Julia Hoeng, Nicolas Sierro, Nikolai V. Ivanov, and Shana J. Sturla
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General Chemical Engineering ,General Chemistry - Abstract
Chemical modifications to DNA bases, including DNA adducts arising from reactions with electrophilic chemicals, are well-known to impact cell growth, miscode during replication, and influence disease etiology. However, knowledge of how genomic sequences and structures influence the accumulation of alkylated DNA bases is not broadly characterized with high resolution, nor have these patterns been linked with overall quantities of modified bases in the genome. For benzo(a) pyrene (BaP), a ubiquitous environmental carcinogen, we developed a single-nucleotide resolution damage sequencing method to map in a human lung cell line the main mutagenic adduct arising from BaP. Furthermore, we combined this analysis with quantitative mass spectrometry to evaluate the dose-response profile of adduct formation. By comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data, we found that although overall adduct levels rose with increasing chemical exposure concentration, genomic distribution patterns consistently correlated with chromatin state and transcriptional status. Moreover, due to the single nucleotide resolution characteristics of this DNA damage map, we could determine preferred DNA triad sequence contexts for alkylation accumulation, revealing a characteristic DNA damage signature. This new BaP damage signature had a profile highly similar to mutational signatures identified previously in lung cancer genomes from smokers. Thus, these data provide insight on how genomic features shape the accumulation of alkylation products in the genome and predictive strategies for linking single-nucleotide resolution in vitro damage maps with human cancer mutations., ACS Central Science, 9 (3), ISSN:2374-7951
- Published
- 2023
- Full Text
- View/download PDF
3. Investigating Nucleosome Accessibility for MNase, FeII Peplomycin, and Duocarmycin B2by Using Capillary Electrophoresis
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Seiichiro Kizaki, Tingting Zou, and Hiroshi Sugiyama
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0301 basic medicine ,biology ,Chemistry ,Organic Chemistry ,Intercalation (chemistry) ,Texas Red ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Capillary electrophoresis ,Duocarmycin B2 ,biology.protein ,Biophysics ,Molecular Medicine ,Nucleosome ,Peplomycin ,Molecular Biology ,DNA ,Micrococcal nuclease - Abstract
Capillary electrophoresis, coupled with DNA 5' Texas Red labeling, was used to investigate the ability of MNase, FeII peplomycin, and duocarmycin B2 to access the nucleosome. Distinct accessibility patterns of these species to the nucleosome were observed. MNase was completely prevented from approaching the nucleosome core and exhibited a higher site specificity for targeting DNA sites located close to the core region. Intercalation of peplomycin in the nucleosomal core region was highly suppressed, but reaction sites located at the ends of the nucleosomal core remained accessible, which implied flexibility of the core DNA end. Duocarmycin B2 was able to enter and react in the core region, although its alkylating efficiency decreased significantly.
- Published
- 2018
4. UVA irradiation of BrU-substituted DNA in the presence of Hoechst 33258
- Author
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Hiroshi Sugiyama, Zutao Yu, Ji Hoon Han, Seiichiro Kizaki, and Abhijit Saha
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0301 basic medicine ,5-Bromouracil ,DNA repair ,Clinical Biochemistry ,Pharmaceutical Science ,Context (language use) ,Biochemistry ,Electron transfer ,03 medical and health sciences ,chemistry.chemical_compound ,Bromouracil ,Drug Discovery ,Nucleosome ,DNA photoreaction ,Molecular Biology ,Micro-irradiation ,030102 biochemistry & molecular biology ,Chemistry ,Organic Chemistry ,Linker DNA ,Molecular biology ,Bisbenzimidazole ,030104 developmental biology ,Biophysics ,Molecular Medicine ,biological phenomena, cell phenomena, and immunity ,DNA - Abstract
Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil (BrU)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to BrU caused DNA cleavage preferentially at self-complementary 5′-AABrUBrU-3′ sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA.
- Published
- 2018
5. Preferential 5-Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein
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Tingting Zou, Yuki Suzuki, Seiichiro Kizaki, Yue Li, Yoshie Harada, Hiroshi Sugiyama, and Yong-Woon Han
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0301 basic medicine ,Stereochemistry ,Catalysis ,law.invention ,Cytosine ,03 medical and health sciences ,chemistry.chemical_compound ,law ,Nucleosome ,AFM imaging ,Histone octamer ,Organic Chemistry ,DNA ,General Chemistry ,DNA Methylation ,Linker DNA ,proteins ,Nucleosomes ,5-Methylcytosine ,030104 developmental biology ,DNA demethylation ,Biochemistry ,chemistry ,Recombinant DNA ,Oxidation-Reduction ,Linker - Abstract
Tet (ten-eleven translocation) family proteins oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG-methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region.
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- 2016
6. Investigating Nucleosome Accessibility for MNase, Fe
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Tingting, Zou, Seiichiro, Kizaki, and Hiroshi, Sugiyama
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Duocarmycins ,Indoles ,Peplomycin ,Electrophoresis, Capillary ,Micrococcal Nuclease ,DNA ,Ferrous Compounds ,DNA Cleavage ,Pyrrolidinones ,Nucleosomes - Abstract
Capillary electrophoresis, coupled with DNA 5' Texas Red labeling, was used to investigate the ability of MNase, Fe
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- 2017
7. Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA
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Yue Li, Fumitaka Hashiya, Hiroshi Sugiyama, Abhijit Saha, and Seiichiro Kizaki
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5-Bromouracil ,Bromouracil ,Sequence analysis ,Ultraviolet Rays ,Uracil ,DNA ,Sequence Analysis, DNA ,Biology ,Medicinal chemistry ,Molecular biology ,chemistry.chemical_compound ,chemistry ,Chemical Biology and Nucleic Acid Chemistry ,Uracil-DNA glycosylase ,Genetics ,Electrophoresis, Polyacrylamide Gel ,Furans ,Uracil-DNA Glycosidase ,Tetrahydrofuran - Abstract
In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil ((Br)U)-substituted sequence 5'-(G/C)[A]n = 1,2 (Br)U(Br)U-3' and proposed that a formed uracil-5-yl radical mainly abstracts the C1' hydrogen from the 5'-side of (Br)U(Br)U under 302-nm irradiation condition. In the present work, we performed photoirradiation of (Br)U-substituted DNA in the presence of a hydrogen donor, tetrahydrofuran, to quench the uracil-5-yl radical to uracil and then subjected the sample to uracil DNA glycosylase digestion. Slab gel sequence analysis indicated that uracil residues were formed at the hot-spot sequence of 5'-(G/C)[A]n = 1,2 (Br)U(Br)U-3' in 302-nm irradiation of (Br)U-substituted DNA. Furthermore, we found that the uracil residue was also formed at the reverse sequence 5'-(Br)U(Br)U[A]n = 1,2(G/C)-3', which suggests that both 5'-(G/C)[A]n = 1,2 (Br)U(Br)U-3' and 5'-(Br)U(Br)U[A]n = 1,2(G/C)-3' are hot-spot sequences for the formation of the uracil-5-yl radical.
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- 2014
8. Advancing Small-Molecule-Based Chemical Biology with Next-Generation Sequencing Technologies
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Ganesh N. Pandian, Chandran Anandhakumar, Hiroshi Sugiyama, Seiichiro Kizaki, and Toshikazu Bando
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aptamers ,Chemical biology ,chemical biology ,Genomics ,Nanotechnology ,Biology ,Biochemistry ,DNA sequencing ,Small Molecule Libraries ,Qualitative analysis ,Drug Discovery ,genomics ,Humans ,Precision Medicine ,Molecular Biology ,Genome, Human ,Scale (chemistry) ,Organic Chemistry ,Computational Biology ,High-Throughput Nucleotide Sequencing ,Data science ,Small molecule ,High-Throughput Screening Assays ,small molecules ,Nucleic Acid Conformation ,Molecular Medicine ,next-generation sequencing - Abstract
Next-generation-sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA-sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in-depth insight into small-molecule-triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small-molecule development and screening.
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- 2014
9. UVA irradiation of
- Author
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Abhijit, Saha, Seiichiro, Kizaki, Ji Hoon, Han, Zutao, Yu, and Hiroshi, Sugiyama
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Bromouracil ,Structure-Activity Relationship ,Dose-Response Relationship, Drug ,Free Radicals ,Molecular Structure ,Ultraviolet Rays ,Bisbenzimidazole ,DNA Breaks, Double-Stranded ,DNA ,DNA Cleavage - Abstract
Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil (
- Published
- 2017
10. Sequence-specific DNA alkylation and transcriptional inhibition by long-chain hairpin pyrrole–imidazole polyamide–chlorambucil conjugates targeting CAG/CTG trinucleotide repeats
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Toshikazu Bando, Kaori Hashiya, Makoto Yamamoto, Fumitaka Hashiya, Sefan Asamitsu, Hiroshi Sugiyama, Seiichiro Kizaki, and Yusuke Kawamoto
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Alkylation ,Transcription, Genetic ,DNA damage ,Clinical Biochemistry ,Pharmaceutical Science ,Biochemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Trinucleotide Repeats ,RNA polymerase ,Drug Discovery ,Peptide synthesis ,Structure–activity relationship ,Pyrroles ,Molecular Biology ,Organic Chemistry ,Imidazoles ,DNA ,Molecular biology ,Nylons ,DNA Alkylation ,chemistry ,Molecular Medicine ,Chlorambucil ,Conjugate - Abstract
Introducing novel building blocks to solid-phase peptide synthesis, we readily synthesized long-chain hairpin pyrrole-imidazole (PI) polyamide-chlorambucil conjugates 3 and 4 via the introduction of an amino group into a GABA (γ-turn) contained in 3, to target CAG/CTG repeat sequences, which are associated with various hereditary disorders. A high-resolution denaturing polyacrylamide sequencing gel revealed sequence-specific alkylation both strands at the N3 of adenines or guanines in CAG/CTG repeats by conjugates 3 and 4, with 11bp recognition. In vitro transcription assays using conjugate 4 revealed that specific alkylation inhibited the progression of RNA polymerase at the alkylating sites. Chiral substitution of the γ-turn with an amino group resulted in higher binding affinity observed in SPR assays. These assays suggest that conjugates 4 with 11bp recognition has the potential to cause specific DNA damage and transcriptional inhibition at the alkylating sites.
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- 2014
11. Sequence-specific electron injection into DNA from an intermolecular electron donor
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Tomohiro Takenaka, Seiichiro Kizaki, Toshikazu Bando, Kaori Hashiya, Fumitaka Hashiya, Hironobu Morinaga, and Hiroshi Sugiyama
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chemistry.chemical_classification ,Bromouracil ,Pyrenes ,Base Sequence ,Imidazoles ,Electrons ,Electron donor ,DNA ,Biology ,Electron acceptor ,Electron transport chain ,Nylons ,chemistry.chemical_compound ,Electron transfer ,chemistry ,Biochemistry ,Structural Biology ,Intramolecular force ,DNA nanotechnology ,Genetics ,Biophysics ,Pyrroles ,Binding site - Abstract
Electron transfer in DNA has been intensively studied to elucidate its biological roles and for applications in bottom-up DNA nanotechnology. Recently, mechanisms of electron transfer to DNA have been investigated; however, most of the systems designed are intramolecular. Here, we synthesized pyrene-conjugated pyrrole-imidazole polyamides (PPIs) to achieve sequence-specific electron injection into DNA in an intermolecular fashion. Electron injection from PPIs into DNA was detected using 5-bromouracil as an electron acceptor. Twelve different 5-bromouracil-containing oligomers were synthesized to examine the electron-injection ability of PPI. Product analysis demonstrated that the electron transfer from PPIs was localized in a range of 8 bp from the binding site of the PPIs. These results demonstrate that PPIs can be a useful tool for sequence-specific electron injection.
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- 2013
12. Photoreactivities of 5-Bromouracil-containing RNAs
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Tomohiro Takenaka, Hironobu Morinaga, Hiroshi Sugiyama, Yuta Sannohe, Ryu Tashiro, Seiichiro Kizaki, and Shuhei Kanesato
- Subjects
5-Bromouracil ,Bromouracil ,Spectrometry, Mass, Electrospray Ionization ,Light ,Stereochemistry ,Clinical Biochemistry ,Pharmaceutical Science ,Guanosine ,Biochemistry ,High-performance liquid chromatography ,Electron transfer ,Photoreaction ,chemistry.chemical_compound ,Drug Discovery ,medicine ,Nucleotide ,Molecular Biology ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Organic Chemistry ,Ms analysis ,RNA ,Adenosine ,chemistry ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,medicine.drug - Abstract
5-Bromouracil ( Br U) was incorporated into three types of synthetic RNA and the products of the photoirradiated Br U-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA Br UGC) 2 and r(CGAA Br UUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2′-keto adenosine ( keto A) product at the 5′-neighboring nucleotide, such as r(GC keto AUGC) and r(CGA keto AUUGC), respectively. The photoirradiation of r(CGCG Br UGCG)/r(C m GCAC m GCG) in Z-form RNA produced the 2′-keto guanosine ( keto G) product r(CGC keto GUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG Br UGCG)/r(C m GCAC m GCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2′ position to provide a 2′-keto RNA product.
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- 2013
13. Nucleosome Assembly Alters the Accessibility of the Antitumor Agent Duocarmycin B2 to Duplex DNA
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Seiichiro Kizaki, Ganesh N. Pandian, Tingting Zou, and Hiroshi Sugiyama
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0301 basic medicine ,Indoles ,Nucleosome assembly ,Alkylation ,Stereochemistry ,Antineoplastic Agents ,010402 general chemistry ,01 natural sciences ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Duocarmycins ,Nucleosome ,Gel electrophoresis ,Base Sequence ,Chemistry ,Organic Chemistry ,General Chemistry ,DNA ,Linker DNA ,In vitro ,Pyrrolidinones ,0104 chemical sciences ,Nucleosomes ,030104 developmental biology ,Naked DNA ,Biophysics - Abstract
To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B2 on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B2 were significantly decreased in core DNA and increased at the histone-free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B2 to duplex DNA could advance its design as an antitumor agent.
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- 2016
14. Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing
- Author
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Seiichiro Kizaki, Hiroshi Sugiyama, and Anandhakumar Chandran
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0301 basic medicine ,Chromosomal translocation ,Biology ,Biochemistry ,DNA sequencing ,Mixed Function Oxygenases ,03 medical and health sciences ,chemistry.chemical_compound ,Data sequences ,Proto-Oncogene Proteins ,Epigenetics ,5-hydroxymethylcytosine ,Molecular Biology ,Sequence (medicine) ,5-Hydroxymethylcytosine ,DNA recognition ,Organic Chemistry ,high-throughput sequencing ,Highly selective ,Molecular biology ,High-Throughput Screening Assays ,5-Methylcytosine ,030104 developmental biology ,chemistry ,Molecular Medicine ,sequence determination ,Oxidation-Reduction ,Tet - Abstract
Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein.
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- 2015
15. Controlling Electron Rebound within Four-Base π-Stacks in Z-DNA by Changing the Sugar Moiety from Deoxy- to Ribonucleotide
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Ayaka Fujiwara, Yuta Sannohe, Yue Li, Hiroshi Sugiyama, Ryu Tashiro, Seiichiro Kizaki, Hironobu Morinaga, and Shuhei Kanesato
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5-Bromouracil ,Bromouracil ,Ribonucleotide ,Ultraviolet Rays ,Organic Chemistry ,Carbohydrates ,Deoxyguanosine ,Guanosine ,Electrons ,General Chemistry ,Electron ,Ribonucleotides ,Photochemistry ,Catalysis ,Electron Transport ,Z-DNA ,chemistry.chemical_compound ,Electron transfer ,chemistry ,DNA, Z-Form ,Quantum Theory ,DNA - Abstract
Charge transfer through DNA is of great interest because of the potential of DNA to be a building block for nanoelectronic sensors and devices. The photochemical reaction of 5-halouracil has been used for probing charge-transfer processes along DNA. We previously reported on unique charge transfer following photochemical reaction of 5-bromouracil within four-base π-stacks in Z-DNA. In this study, we incorporated a guanosine instead of a deoxyguanosine into Z-DNA, and found that electron transfer occurs in a different mechanism through four-base π-stacks.
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- 2013
16. A novel detection technique of polyamide binding sites by photo-induced electron transfer in (Br)U substituted DNA
- Author
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Kaori Hashiya, Toshikazu Bando, Seiichiro Kizaki, Abhijit Saha, Fumitaka Hashiya, Sefan Asamitsu, and Hiroshi Sugiyama
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Bromouracil ,Binding Sites ,fungi ,Metals and Alloys ,Electrons ,General Chemistry ,DNA ,Conjugated system ,Photochemistry ,Combinatorial chemistry ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,chemistry.chemical_compound ,Electron transfer ,Nylons ,chemistry ,Polyamide ,Materials Chemistry ,Ceramics and Composites ,Pyrene ,Imidazole ,Binding site ,Pyrrole - Abstract
We report the photochemistry of (Br)U substituted DNA as a versatile platform to investigate the binding sites of pyrene conjugated pyrrole imidazole polyamides (PIPs). The results suggest that the approach can be used on a routine basis for the screening of polyamide binding sites.
- Published
- 2015
17. Detection of UV-induced cyclobutane pyrimidine dimers by near-infrared spectroscopy and aquaphotomics
- Author
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Makoto Kunisada, Roumiana Tsenkova, György Bázár, Seiichiro Kizaki, Noriko Goto, Hiroshi Sugiyama, Zoltan Kovacs, Chikako Nishigori, and Hiroyuki Morita
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Multidisciplinary ,Spectroscopy, Near-Infrared ,DNA damage ,Chemistry ,Ultraviolet Rays ,Analytical chemistry ,Pyrimidine dimer ,DNA Solutions ,DNA ,medicine.disease_cause ,Article ,Cyclobutane ,chemistry.chemical_compound ,Mutagenesis ,Pyrimidine Dimers ,Partial least squares regression ,Mutation ,medicine ,Spectroscopy ,Ultraviolet ,DNA Damage - Abstract
Ultraviolet (UV) radiation causes cellular DNA damage, among which cyclobutane pyrimidine dimers (CPDs) are responsible for a variety of genetic mutations. Although several approaches have been developed for detection of CPDs, conventional methods require time-consuming steps. Aquaphotomics, a new approach based on near-infrared spectroscopy (NIRS) and multivariate analysis that determines interactions between water and other components of the solution, has become an effective method for qualitative and quantitative parameters measurement in the solutions. NIR spectral patterns of UVC-irradiated and nonirradiated DNA solutions were evaluated using aquaphotomics for detection of UV-induced CPDs. Groups of UV-irradiated and nonirradiated DNA samples were classified (87.5% accuracy) by soft independent modeling of class analogy (SIMCA). A precise regression model calculated from NIR water spectral patterns based on UVC doses (r Val = 0.9457) and the concentration of cis-syn cyclobutane thymine dimers (cis-syn T<>Ts; r Val = 0.9993) was developed using partial least squares regression (PLSR), while taking advantage of water spectral patterns, particularly around 1400–1500 nm. Our results suggested that, in contrast to DNA, the formation of cis-syn T<>Ts increased the strongly hydrogen bonded water. Additionally, NIRS could qualitatively and quantitatively detect cis-syn T<>Ts in isolated DNA aqueous solutions upon UVC exposure.
- Published
- 2015
18. Next-generation sequencing studies guide the design of pyrrole-imidazole polyamides with improved binding specificity by the addition of β-alanine
- Author
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Chandran Anandhakumar, Toshikazu Bando, Yue Li, Hiroshi Sugiyama, Ganesh N. Pandian, Kaori Hashiya, and Seiichiro Kizaki
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musculoskeletal diseases ,High-throughput screening ,Biology ,Biochemistry ,Genome ,DNA sequencing ,chemistry.chemical_compound ,parasitic diseases ,Imidazole ,Pyrroles ,Binding site ,Molecular Biology ,Binding selectivity ,Alanine ,next generation sequencing ,DNA recognition ,Binding Sites ,Base Sequence ,Organic Chemistry ,fungi ,Imidazoles ,High-Throughput Nucleotide Sequencing ,DNA ,Molecular biology ,Small molecule ,body regions ,Nylons ,chemistry ,beta-Alanine ,Molecular Medicine ,lipids (amino acids, peptides, and proteins) ,synthetic biology ,high throughput screening - Abstract
The identification of binding sites for small molecules in genomic DNA is important in various applications. Previously, we demonstrated rapid transcriptional activation by our small molecule SAHA-PIP. However, it was not clear whether the strong biological effects exerted by SAHA-PIP were attributable to its binding specificity. Here, we used high-throughput sequencing (Bind-n-seq) to determine the binding specificity of SAHA-PIPs. Sequence specificity bias was determined for SAHA-PIPs (3 and 4), and this showed enhanced 6 bp sequence-specific binding compared with hairpin PIPs (1 and 2). This finding allowed us to investigate the role of the β-alanine that links SAHA to PIP, and led in turn to the design of ββ-PIPs (5 and 6), which showed enhanced binding specificity. Overall, we demonstrated the importance of β-moieties for the binding specificity of PIPs and the use of cost-effective high-throughput screening of these small molecules for binding to the DNA minor groove.
- Published
- 2014
19. CGmCGCG is a versatile substrate with which to evaluate Tet protein activity
- Author
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Hiroshi Sugiyama and Seiichiro Kizaki
- Subjects
Hplc analysis ,Oligoribonucleotides ,Organic Chemistry ,Substrate (chemistry) ,Oxidation reduction ,biochemical phenomena, metabolism, and nutrition ,Biochemistry ,DNA-binding protein ,Molecular biology ,DNA-Binding Proteins ,5-Methylcytosine ,chemistry.chemical_compound ,chemistry ,stomatognathic system ,polycyclic compounds ,Protein activity ,Physical and Theoretical Chemistry ,Oxidation-Reduction ,DNA - Abstract
Tet family proteins have the ability to convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine, and further to 5-formylcytosine and 5-carboxycytosine. We found that CGmCGCG can be the substrate of Tet protein, and observed iterative oxidation of mC by HPLC analysis. We also demonstrated that Tet protein favours single-stranded DNA over double-stranded DNA.
- Published
- 2013
20. Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay
- Author
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Debojyoti De, Abhijit Saha, Kyeong Kyu Kim, Masayuki Endo, Seiichiro Kizaki, and Hiroshi Sugiyama
- Subjects
0301 basic medicine ,Bromouracil ,Light ,PAX6 Transcription Factor ,genetic structures ,Guanine ,Electrons ,Cooperativity ,Biology ,Protein Structure, Secondary ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Electron transfer ,Protein Domains ,Genetics ,Humans ,DNA Cleavage ,Enhancer ,Uridine ,Transcription factor ,Hypoxanthine ,Base Sequence ,SOXB1 Transcription Factors ,Tryptophan ,Reproducibility of Results ,Cooperative binding ,DNA ,Enhancer Elements, Genetic ,Spectrometry, Fluorescence ,030104 developmental biology ,Biochemistry ,chemistry ,Transcription preinitiation complex ,Biophysics ,Methods Online ,sense organs ,Protein Binding - Abstract
Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, (Br)U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.
- Published
- 2016
21. Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.
- Author
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Saha, Abhijit, Seiichiro Kizaki, De, Debojyoti, Masayuki Endo, Kyeong Kyu Kim, and Hiroshi Sugiyama
- Published
- 2016
- Full Text
- View/download PDF
22. Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA.
- Author
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Fumitaka Hashiya, Saha, Abhijit, Seiichiro Kizaki, Yue Li, and Hiroshi Sugiyama
- Published
- 2014
- Full Text
- View/download PDF
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