Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Yoshizumi Ishino Last modified date:2021.06.16

Professor / Molecular Biosciences / Department of Bioscience and Biotechnology / Faculty of Agriculture

1. Mayanagi, K., Oki, K., Miyazaki, N., Ishino, S., Yamagami, T., Morikawa, K., Iwasaki, K., Kohda, D., Shirai, T., and Ishino, Y., Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy, BMC BIOLOGY, 10.1186/s12915-020-00889-y, 18, 1, 2020.10.
2. Koonin, E.V., Krupovic, M., Ishino, S., and Ishino, Y., The replication machinery of LUCA: common origin of DNA replication and transcription, BMC BIOLOGY, 10.1186/s12915-020-00800-9, 18, 1, 2020.06.
3. Hogrel, G., Lu, Y., Alexandre, N., Bossé, A., Dulermo, R., Ishino, S., Ishino, Y., and Flament, D. , Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea, BIOMOLECULES, 10.3390/biom10071045, 10, 7, 2020.07.
4. Ishino, Y., Studies on DNA-related enzymes to elucidate molecular mechanisms underlying genetic information processing and their application in genetic engineering., Biosci Biotechnol Biochem., 10.1080/09168451.2020.1778441, 1-18. (AWARD REVIEW), 2020.04.
5. Kei Maruyama, Hirotatsu Imai, Momoko Kawamura, Sonoko Ishino, Yoshizumi Ishino, Kosuke Ito, Toshio Uchiumi, Switch of the interactions between the ribosomal stalk and EF1A in the GTP- and GDP-bound conformations, Scientific reports, 10.1038/s41598-019-51266-x, 9, 1, 2019.12, Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal “stalk” protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Å resolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis..
6. Kushida, T., Narumi, I., Ishino, S., Ishino, Y., Fujiwara, S., Imanaka, T., and Higashibata, H. , PolB, a Family B DNA Polymerase, in Thermococcus kodakarensis is important for DNA repair, but not DNA Replication. , Microbes Environ., 10.1264/jsme2.ME19075., 34, 316-326., 2019.07.
7. Yoji Igarashi, Daisuke Mori, Susumu Mitsuyama, Kazutoshi Yoshitake, Hiroaki Ono, Tsuyoshi Watanabe, Yukiko Taniuchi, Tomoko Sakami, Akira Kuwata, Takanori Kobayashi, Yoshizumi Ishino, Shugo Watabe, Takashi Gojobori, Shuichi Asakawa, A preliminary metagenome analysis based on a combination of protein domains, Proteomes, 10.3390/proteomes7020019, 7, 2, 2019.06, Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have not often been fully analyzed through a homology search because the genomic data in databases for living organisms on earth are insufficient. In order to complement the results obtained through homology-search-based methods with shotgun metagenomes data, we focused on the composition of protein domains deduced from the sequences of genomes and metagenomes, and we utilized them in characterizing genomes and metagenomes, respectively. First, we compared the relationships based on similarities in the protein domain composition with the relationships based on sequence similarities. We searched for protein domains of 325 bacterial species produced using the Pfam database. Next, the correlation coefficients of protein domain compositions between every pair of bacteria were examined. Every pairwise genetic distance was also calculated from 16S rRNA or DNA gyrase subunit B. We compared the results of these methods and found a moderate correlation between them. Essentially, the same results were obtained when we used partial random 100 bp DNA sequences of the bacterial genomes, which simulated raw sequence data obtained from short-read next-generation sequences. Then, we applied the method for analyzing the actual environmental data obtained by shotgun sequencing. We found that the transition of the microbial phase occurred because the seasonal change in water temperature was shown by the method. These results showed the usability of the method in characterizing metagenomic data based on protein domain compositions..
8. Natsuki Takashima, Sonoko Ishino, Keisuke Oki, Mika Takafuji, Takeshi Yamagami, Ryotaro Matsuo, Kouta Mayanagi, Yoshizumi Ishino, Elucidating functions of DP1 and DP2 subunits from the Thermococcus kodakarensis family D DNA polymerase, Extremophiles, 10.1007/s00792-018-1070-3, 23, 1, 161-172, 2019.01, DNA polymerase D (PolD), originally discovered in Pyrococcus furiosus, has no sequence homology with any other DNA polymerase family. Genes encoding PolD are found in most of archaea, except for those archaea in the Crenarchaeota phylum. PolD is composed of two proteins: DP1 and DP2. To date, the 3D structure of the PolD heteromeric complex is yet to be determined. In this study, we established a method that prepared highly purified PolD from Thermococcus kodakarensis, and purified DP1 and DP2 proteins formed a stable complex in solution. An intrinsically disordered region was identified in the N-terminal region of DP1, but the static light scattering analysis provided a reasonable molecular weight of DP1. In addition, PolD forms as a complex of DP1 and DP2 in a 1:1 ratio. Electron microscope single particle analysis supported this composition of PolD. Both proteins play an important role in DNA synthesis activity and in 3′–5′ degradation activity. DP1 has extremely low affinity for DNA, while DP2 is mainly responsible for DNA binding. Our work will provide insight and the means to further understand PolD structure and the molecular mechanism of this archaea-specific DNA polymerase..
9. Ying Liu, David Brandt, Sonoko Ishino, Yoshizumi Ishino, Eugene V. Koonin, Jörn Kalinowski, Mart Krupovic, David Prangishvili, New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures, Environmental Microbiology, 10.1111/1462-2920.14479, 2019.01, Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments..
10. Mariko Nagata, Sonoko Ishino, Takeshi Yamagami, Yoshizumi Ishino, Replication protein A complex in Thermococcus kodakarensis interacts with DNA polymerases and helps their effective strand synthesis, Bioscience, Biotechnology and Biochemistry, 10.1080/09168451.2018.1559722, 83, 4, 695-704, 2019.01, Replication protein A (RPA) is an essential component of DNA metabolic processes. RPA binds to single-stranded DNA (ssDNA) and interacts with multiple DNA-binding proteins. In this study, we showed that two DNA polymerases, PolB and PolD, from the hyperthermophilic archaeon Thermococcus kodakarensis interact directly with RPA in vitro. RPA was expected to play a role in resolving the secondary structure, which may stop the DNA synthesis reaction, in the template ssDNA. Our in vitro DNA synthesis assay showed that the pausing was resolved by RPA for both PolB and PolD. These results supported the fact that RPA interacts with DNA polymerases as a member of the replisome and is involved in the normal progression of DNA replication forks..
11. Takashi Kushida, Issay Narumi, Sonoko Ishino, Yoshizumi Ishino, Shinsuke Fujiwara, Tadayuki Imanaka, Hiroki Higashibata, Pol B, a family B DNA polymerase, in Thermococcus kodakarensis is important for DNA repair, but not DNA replication, Microbes and Environments, 10.1264/jsme2.ME19075, 34, 3, 316-326, 2019.01, Thermococcus kodakarensis possesses two DNA polymerases, Pol B and Pol D. We generated a T. kodakarensis strain (DPB1) in which polB was completely deleted and a derivative of DPB1 in which polB was overexpressed; neither of the generated strains exhibited any growth delay, indicating that the lack or overexpression of Pol B in T. kodakarensis did not affect cell growth. We also found that DPB1 showed higher sensitivity to four DNA-damaging agents (ultraviolet C irradiation, γ-ray irradiation, methyl methanesulfonate, and mitomycin C) than the parental strain. The sensitivity of DPB1 was restored to the level of the parent strain by the introduction of a plasmid harboring polB, suggesting that the DNA damage-sensitive phenotype of DPB1 was due to the loss of polB. Collectively, these results indicate that Pol B is involved in DNA repair, but not DNA replication, which, in turn, implies that Pol D is the sole replicative DNA polymerase in Thermococcus species..
12. Kouta Mayanagi, Sonoko Ishino, Tsuyoshi Shirai, Takuji Oyama, Shinichi Kiyonari, Daisuke Kohda, Kosuke Morikawa, Yoshizumi Ishino, Direct visualization of DNA baton pass between replication factors bound to PCNA, Scientific reports, 10.1038/s41598-018-34176-2, 8, 1, 2018.12, In Eukarya and Archaea, the lagging strand synthesis is accomplished mainly by three key factors, DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig), in the DNA replication process. These three factors form important complexes with proliferating cell nuclear antigen (PCNA), thereby constructing a platform that enable each protein factor to act successively and smoothly on DNA. The structures of the Pol-PCNA-DNA and Lig-PCNA-DNA complexes alone have been visualized by single particle analysis. However, the FEN-PCNA-DNA complex structure remains unknown. In this report, we for the first time present this tertiary structure determined by single particle analysis. We also successfully visualized the structure of the FEN-Lig-PCNA-DNA complex, corresponding to a putative intermediate state between the removal of the DNA flap by FEN and the sealing of the nicked DNA by Lig. This structural study presents the direct visualization of the handing-over action, which proceeds between different replication factors on a single PCNA clamp bound to DNA. We detected a drastic conversion of the DNA from a bent form to a straight form, in addition to the dynamic motions of replication factors in the switching process..
13. Miyako Shiraishi, Sonoko Ishino, Matthew Heffernan, Isaac Cann, Yoshizumi Ishino, The mesophilic archaeon Methanosarcina acetivorans counteracts uracil in DNA with multiple enzymes
EndoQ, ExoIII, and UDG, Scientific reports, 10.1038/s41598-018-34000-x, 8, 1, 2018.12, Cytosine deamination into uracil is one of the most prevalent and pro-mutagenic forms of damage to DNA. Base excision repair is a well-known process of uracil removal in DNA, which is achieved by uracil DNA glycosylase (UDG) that is found in all three domains of life. However, other strategies for uracil removal seem to have been evolved in Archaea. Exonuclease III (ExoIII) from the euryarchaeon Methanothermobacter thermautotrophicus has been described to exhibit endonuclease activity toward uracil-containing DNA. Another uracil-acting protein, endonuclease Q (EndoQ), was recently identified from the euryarchaeon Pyrococcus furiosus. Here, we describe the uracil-counteracting system in the mesophilic euryarchaeon Methanosarcina acetivorans through genomic sequence analyses and biochemical characterizations. Three enzymes, UDG, ExoIII, and EndoQ, from M. acetivorans exhibited uracil cleavage activities in DNA with a distinct range of substrate specificities in vitro, and the transcripts for these three enzymes were detected in the M. acetivorans cells. Thus, this organism appears to conduct uracil repair using at least three distinct pathways. Distribution of the homologs of these uracil-targeting proteins in Archaea showed that this tendency is not restricted to M. acetivorans, but is prevalent and diverse in most Archaea. This work further underscores the importance of uracil-removal systems to maintain genome integrity in Archaea, including ‘UDG lacking’ organisms..
14. Sonoko Ishino, Stéphane Skouloubris, Hanae Kudo, Caroline L'Hermitte-Stead, Asmae Es-Sadik, Jean Christophe Lambry, Yoshizumi Ishino, Hannu Myllykallio, Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication, Nucleic acids research, 10.1093/nar/gky460, 46, 12, 6206-6217, 2018.07, The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the β-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-β'clamp complex formed via a carboxy-terminal sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system..
15. Miyazono, K.I., Ishino, S., Makita, N., Ito, T., Ishino, Y., and Tanokura, M., Crystal structure of the novel lesion-specific endonuclease PfuEndoQ from Pyrococcus furiosus., Nucleic Acids Res., doi: 10.1093/nar/gky261., 46, 9, 4807-4818, 2018.05, Because base deaminations, which are promoted by high temperature, ionizing radiation, aerobic respiration and nitrosative stress, produce mutations during replication, deaminated bases must be repaired quickly to maintain genome integrity. Recently, we identified a novel lesion-specific endonuclease, PfuEndoQ, from Pyrococcus furiosus, and PfuEndoQ may be involved in the DNA repair pathway in Thermococcales of Archaea. PfuEndoQ recognizes a deaminated base and cleaves the phosphodiester bond 5' of the lesion site. To elucidate the structural basis of the substrate recognition and DNA cleavage mechanisms of PfuEndoQ, we determined the structure of PfuEndoQ using X-ray crystallography. The PfuEndoQ structure and the accompanying biochemical data suggest that PfuEndoQ recognizes a deaminated base using a highly conserved pocket adjacent to a Zn2+-binding site and hydrolyses a phosphodiester bond using two Zn2+ ions. The PfuEndoQ-DNA complex is stabilized by a Zn-binding domain and a C-terminal helical domain, and the complex may recruit downstream proteins in the DNA repair pathway..
16. Yoshizumi Ishino, Mart Krupovic, Patrick Forterre, History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology, Journal of bacteriology, 10.1128/JB.00580-17, 200, 7, 2018.04, Clustered regularly interspaced short palindromic repeat (CRISPR)- Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in the Escherichia coli genome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence in E. coli to genome editing in humans..
17. Katsuya Daimon, Sonoko Ishino, Namiko Imai, Sachiyo Nagumo, Takeshi Yamagami, Hiroaki Matsukawa, Yoshizumi Ishino, Two family B DNA polymerases from Aeropyrum pernix, based on revised translational frames, Frontiers in Molecular Biosciences, 10.3389/fmolb.2018.00037, 5, APR, 2018.04, Living organisms are divided into three domains, Bacteria, Eukarya, and Archaea. Comparative studies in the three domains have provided useful information to understand the evolution of the DNA replication machinery. DNA polymerase is the central enzyme of DNA replication. The presence of multiple family B DNA polymerases is unique in Crenarchaeota, as compared with other archaeal phyla, which have a single enzyme each for family B (PolB) and family D (PolD). We analyzed PolB1 and PolB3 in the hyperthermophilic crenarchaeon, Aeropyrum pernix, and found that they are larger proteins than those predicted from the coding regions in our previous study and from public database annotations. The recombinant larger PolBs exhibited the same DNA polymerase activities as previously reported. However, the larger PolB3 showed remarkably higher thermostability, which made this enzyme applicable to PCR. In addition, the high tolerance to salt and heparin suggests that PolB3 will be useful for amplification from the samples with contaminants, and therefore it has a great potential for diagnostic use in the medical and environmental field..
18. Nagata, M., Ishino, S., Yamagami, T., Simons, J-R., Kanai, T., Atomi, H., and Ishino, Y. , Possible function of the second RecJ-like protein in stalled replication fork repair by interacting with Hef. , Sci Rep. , doi: 10.1038/s41598-017-17306-0., 7, 1, 16949, 2017.12, RecJ was originally identified in Escherichia coli and plays an important role in the DNA repair and recombination pathways. Thermococcus kodakarensis, a hyperthermophilic archaeon, has two RecJ-like nucleases. These proteins are designated as GAN (GINS-associated nuclease) and HAN (Hef-associated nuclease), based on the protein they interact with. GAN is probably a counterpart of Cdc45 in the eukaryotic CMG replicative helicase complex. HAN is considered mainly to function with Hef for restoration of the stalled replication fork. In this study, we characterized HAN to clarify its functions in Thermococcus cells. HAN showed single-strand specific 3' to 5' exonuclease activity, which was stimulated in the presence of Hef. A gene disruption analysis revealed that HAN was non-essential for viability, but the ΔganΔhan double mutant did not grow under optimal conditions at 85 °C. This deficiency was not fully recovered by introducing the mutant han gene, encoding the nuclease-deficient HAN protein, back into the genome. These results suggest that the unstable replicative helicase complex without GAN performs ineffective fork progression, and thus the stalled fork repair system including HAN becomes more important. The nuclease activity of HAN is required for the function of this protein in T. kodakarensis..
19. Nagata, M., Ishino, S., Yamagami, T., Ogino, H., Simons, J-R., Kanai, T., Atomi, H., and Ishino, Y. , The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis. , Nucleic Acids Res., doi: 10.1093/nar/gkx740., 45, 18, 10693. -10705. , 2017.10, The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells.

20. Antranikian, G., Suleiman, M., Schafers, C., Adams, MWW., Bartolucci, S., Blamey, JM., Birkeland, NK., Bonch-Osmolovskaya, E., da Costa, MS., Cowan, D., Danson, M., Forterre, P., Kelly, R., Ishino, Y., Littlechild, J., Moracci, M., Noll, K., Oshima, T., Robb, F., Rossi, M., Santos, H., Schonheit, P., Sterner, R., Thauer, R.,Thomm, M., Wiegel, J., and Stetter, KO., Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island., Extremophiles., doi: 10.1007/s00792-017-0938-y., 21, 733.-742., 2017.07, To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80 °C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100 °C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, ε-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature..
21. Liu, S., Ishino, S., Ishino, Y., Pehau-Arnaudet, G., Krupovic, M., and Prangishvili, D. , A novel type of polyhedral viruses infecting hyperthermophilic archaea., J. Virol. , doi: 10.1128/JVI.00589-17., 91, e00589-17. , 2017.06, Encapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known viruses. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20-kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales The SPV1 genome consists of 20,222 bp and contains 45 open reading frames, only one-fifth of which could be functionally annotated.IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered a representative of a new viral family, "Portogloboviridae.".
22. Tsuchida, N., Ikeda, M., Ishino, Y., Grieco, M., and Vecchio, G., FUCA1 is induced by wild-type p53 and expressed at different levels in thyroid cancers depending on p53 status., Int. J. Oncol. , doi: 10.3892/ijo.2017.3968. , 50, 2043. -2048. , 2017.06, Fucose residues of cell surface glycans, which play important roles in growth, invasion and metastasis, are added by fucosyltransferases (FUTs) and removed by α-L-fucosidases (FUCAs). By the differential display method, we isolated a 3' non-coding region of α-L-fucosidase-1 (FUCA1) (a gene coding for the lysosomal fucosidase-1 enzyme) as a wild-type p53-inducible gene: 18S and 20S FUCA1 mRNA species were induced in Saos-2 cells transfected with a temperature-sensitive p53 mutant at the permissive temperature. By microarray analyses of thyroid cancer biopsy samples, FUCA1 RNA expression levels were found to be lower in anaplastic thyroid cancer samples (ATCs), while they were higher in papillary thyroid cancer samples (PTCs) and in normal thyroid tissues. Since most ATCs were reported to carry the mutated form of p53, while PTCs carry mostly the wild-type form of p53, it is likely that FUCA1 expression levels are regulated, at least in part, by the p53 status in thyroid cancers. In order to better understand the role played by FUCA genes in thyroid tumorigenesis, we examined the clonogenic potential in vitro of thyroid cell lines transfected with either FUCA1 or FUCA2 (the latter gene coding for a secreted, non-lysosomal enzyme). We found that α-L-fucosidases did not suppress grossly cell growth. Contrary to what we observed with the expression of FUCA1, the FUT8 expression levels were found high in ATCs but lower in PTCs and normal thyroid tissues. Taken together, these results suggest the possibility that the higher fucose levels on cell surface glycans of aggressive ATCs, compared to those of less aggressive PTCs, may be at least in part responsible for the more aggressive and metastatic phenotype of ATCs compared to PTCs, as the expression levels of FUCA1 and FUT8 were inversely related in these two types of cancers..
23. Yoshizumi Ishino, Sonoko Ishino, Takeshi YAMAGAMI, Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication. , Nucleic Acids Res., doi: 10.1093/nar/gkw789 , 44, 19, 9509-9517, 2017.05, In eukaryotic DNA replication initiation, hexameric
MCM (mini-chromosome maintenance) unwinds the
template double-stranded DNA to form the replication
fork. MCM is activated by two proteins, Cdc45
and GINS, which constitute the ‘CMG’ unwindosome
complex together with the MCM core. The archaeal
DNA replication system is quite similar to that of
eukaryotes, but only limited knowledge about the
DNA unwinding mechanism is available, froma structural
point of view. Here, we describe the crystal
structure of an archaeal GAN (GINS-associated nuclease)
from Thermococcus kodakaraensis, the homolog
of eukaryotic Cdc45, in both the free form and
the complex with the C-terminal domain of the cognate
Gins51 subunit (Gins51C). This first archaeal
GAN structure exhibits a unique, ‘hybrid’ structure
between the bacterial RecJ and the eukaryotic Cdc45.
GAN possesses the conserved DHH and DHH1 domains
responsible for the exonuclease activity, and
an inserted CID (CMG interacting domain)-like domain
structurally comparable to that in Cdc45, suggesting
its dual roles as an exonuclease in DNA repair
and a CMG component in DNA replication. A
structural comparison of the GAN–Gins51C complex
with the GINS tetramer suggests that GINS uses the
mobile Gins51C as a hook to bind GAN for CMG formation.
DNA replication is essential for all living organisms, and
the basic mechanism is conserved across the three domains
of life, Bacteria, Archaea, and Eukarya. DNA replication
must occur accurately in a highly coordinated manner regulated
by numerous proteins (1). At the initiation of DNA
replication, the parental double-stranded DNA (dsDNA)
is unwound to generate two single-stranded DNAs (ssDNAs),
which form a replication fork. In eukaryotes, the
hetero-hexameric MCM (mini-chromosome maintenance)
comprisingMCM2–7acts as the helicase core to unwind the
templateDNA (2,3). Although isolatedMCMgenerally exhibits
weak helicase activity, two protein factors, Cdc45 and
GINS, have been identified as MCM activators, and they
form the CMG complex holoenzyme together with MCM
(4,5). CMG is constructed on the DNA by the sequential
loading of MCM (as the double-hexameric ring at the formation
of Pre-RC; pre-replicating complex), Cdc45 (at the
formation of Pre-IC; pre-initiation complex) and GINS (at
the Pre-LC; pre-loading complex after pre-IC), rather than
the loading of the pre-assembled complex (3,6,7).CMGformation
is controlled by two protein kinases, CDK (cyclindependent
kinase) and DDK (dbf4-dependent kinase), and
24. Ogino, H., Ishino, S., Kohda, D., and Ishino, Y., The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3'-5' exonuclease and associates with a DNA replication complex., J. Biol. Chem., doi: 10.1074/jbc.M116.767921., 292, 7921.-7931., 2017.05, RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e. in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in the CMG complex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11-3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN (GINS-associated nuclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM (T. acidophilum MCM) helicase in vitro, and the TaRecJ2·TaMCM·TaGINS complex was also observed in vivo However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya..
25. Yoda, T., Tanabe, M., Tsuji, T.,Yoda, T., Ishino, S., Shirai, T., Ishino, Y., Takeyama, H., and Nishida, H. , Exonuclease processivity of archaeal replicative DNA polymerase in association with PCNA is expedited by mismatches in DNA. , Sci. Rep. , doi: 10.1038/srep44582., 7, 44582, 2017.03, Family B DNA polymerases comprise polymerase and 3' ->5' exonuclease domains, and detect a mismatch in a newly synthesized strand to remove it in cooperation with Proliferating cell nuclear antigen (PCNA), which encircles the DNA to provide a molecular platform for efficient protein-protein and protein-DNA interactions during DNA replication and repair. Once the repair is completed, the enzyme must stop the exonucleolytic process and switch to the polymerase mode. However, the cue to stop the degradation is unclear. We constructed several PCNA mutants and found that the exonuclease reaction was enhanced in the mutants lacking the conserved basic patch, located on the inside surface of PCNA. These mutants may mimic the Pol/PCNA complex processing the mismatched DNA, in which PCNA cannot interact rigidly with the irregularly distributed phosphate groups outside the dsDNA. Indeed, the exonuclease reaction with the wild type PCNA was facilitated by mismatched DNA substrates. PCNA may suppress the exonuclease reaction after the removal of the mismatched nucleotide. PCNA seems to act as a "brake" that stops the exonuclease mode of the DNA polymerase after the removal of a mismatched nucleotide from the substrate DNA, for the prompt switch to the DNA polymerase mode..
26. Yoshizumi Ishino, Sonoko Ishino, A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus., Biosci. Biotechnol. Biochem. 81, 931-937. doi: 10.1080/09168451.2016.1277946., doi: 10.1080/09168451.2016.1277946., 81, 931-937., 2017.01, DNA base deamination occurs spontaneously
under physiological conditions and is promoted by
high temperature. Therefore, hyperthermophiles are
expected to have efficient repair systems of the
deaminated bases in their genomes. Endonuclease Q
(EndoQ) was originally identified from the hyperthermophlic
archaeon, Pyrococcus furiosus, as a
hypoxanthine-specific endonuclease recently. Further
biochemical analyses revealed that EndoQ also recognizes
uracil, xanthine, and the AP site in DNA,
and is probably involved in a specific repair process
for damaged bases. Initial phylogenetic analysis
showed that an EndoQ homolog is found only in the
Thermococcales and some of the methanogens in
Archaea, and is not present in most members of the
domains Bacteria and Eukarya. A better understanding
of the distribution of the EndoQ-mediated
repair system is, therefore, of evolutionary interest.
We showed here that an EndoQ-like polypeptide
from Bacillus pumilus, belonging to the bacterial
domain, is functional and has similar properties
with the archaeal EndoQs..
27. Yoshizumi Ishino, Pyrococcus furious単離30周年に寄せて 極限環境生物学会雑誌 15, 5-9., 極限環境生物学誌, 15, 5-9., 5-9., 2016.12.
28. Yoshizumi Ishino, Sonoko Ishino, Structure of the EndoMS-DNA complex as mismatch-restriction endonuclease. Structure 24, 1960-1971. doi: 10.1016/j.str.2016.09.005., Structure,, 24, 1960-1971, 2016.11, Archaeal NucS nuclease was thought to degrade
the single-stranded region of branched DNA, which
contains flapped and splayed DNA. However,
recent findings indicated that EndoMS, the orthologous
enzyme of NucS, specifically cleaves doublestranded
DNA (dsDNA) containing mismatched
bases. In this study, we determined the structure of
the EndoMS-DNA complex. The complex structure
of the EndoMS dimer with dsDNA unexpectedly revealed
that the mismatched bases were flipped out
into binding sites, and the overall architecture most
resembled that of restriction enzymes. The structure
of the apo form was similar to the reported structure
of Pyrococcus abyssi NucS, indicating that movement
of the C-terminal domain from the resting state
was required for activity. In addition, a model of
the EndoMS-PCNA-DNA complex was preliminarily
verified with electron microscopy. The structures
strongly support the idea that EndoMS acts in a
mismatch repair pathway..
29. Yoshizumi Ishino, 細菌に存在する繰り返し配列の発見からゲノム編集技術へ
, 大阪大学医学部学友会会誌 , 36, 54-59, 2016.11, 今から30年前、筆者が大阪大学微生物病研究所において、大腸菌から奇妙な繰り返し配列を発見した時、それが何を意味するのかまったく不明であったが、あまりに特徴的な配列であったことが印象深い。今世紀に入ってから, それはCRISPR (clustered regularly interspaced short palindromic repeats) と呼ばれるようになり、真正細菌, アーキアにとっての獲得免疫機能を担っていることが分かった。人類はこの原核生物の獲得免疫の仕組みを、生物のゲノムDNAを狙ったところで切断して、その位置の遺伝子を破壊したり改変したりする技術へ応用することを思いついた。すなわち、CRISPRの応用によって人類は望んでいた簡便で実用的なゲノム編集技術を手に入れたのである。本稿では大腸菌の基礎研究から思いがけなく発見された繰り返し配列が、後にCRISPRとして有名になるまでを紹介する。.
30. Yoshizumi Ishino, Sonoko Ishino, DJ-1 family Maillard deglycases prevent acrylamide formation. Biochem Biophys Res Commun., Biochem Biophys Res Commun.,, 478, 1111-1116, 2016.08, The presence of acrylamide in food is a worldwide concern because it is carcinogenic, reprotoxic and
neurotoxic. Acrylamide is generated in the Maillard reaction via condensation of reducing sugars and
glyoxals arising from their decomposition, with asparagine, the amino acid forming the backbone of the
acrylamide molecule. We reported recently the discovery of the Maillard deglycases (DJ-1/Park7 and its
prokaryotic homologs) which degrade Maillard adducts formed between glyoxals and lysine or arginine
amino groups, and prevent glycation damage in proteins. Here, we show that these deglycases prevent
acrylamide formation, likely by degrading asparagine/glyoxal Maillard adducts. We also report the discovery
of a deglycase from the hyperthermophilic archaea Pyrococcus furiosus, which prevents acrylamide
formation at 100 C. Thus, Maillard deglycases constitute a unique enzymatic method to prevent
acrylamide formation in food without depleting the components (asparagine and sugars) responsible for
its formation..
31. Yoshizumi Ishino, Sonoko Ishino, Archaeal DNA polymerase-B as a DNA template guardian: links between polymerases and base/alternative excision repair enzymes in handling the deaminated bases uracil and hypoxanthine., Archaea,, 2016, Article ID 1510938, 8 pages, 2016.08, In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three
enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil); Endonuclease V (EndoV, which recognises hypoxanthine); and
Endonuclease Q (EndoQ), (which recognises both uracil and hypoxanthine). Two archaeal DNA polymerases, Pol-B and Pol-D,
are inhibited by deaminated bases in template strands, a feature unique to this domain.Thus the three repair enzymes and the two
polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primertemplates
containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns
off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be
a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the
presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting
as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNAmutations..
32. Yoshizumi Ishino, Sonoko Ishino, Takeshi YAMAGAMI, PCNA is involved in the EndoQ-mediated DNA repair process in Thermococcales. , Scientific Report, DOI: 10.1038/srep25532, 6, 25532, 2016.05, To maintain genome integrity for transfer to their offspring, and to maintain order in cellular processes,
all living organisms have DNA repair systems. Besides the well-conserved DNA repair machineries,
organisms thriving in extreme environments are expected to have developed efficient repair
systems. We recently discovered a novel endonuclease, which cleaves the 5′ side of deoxyinosine,
from the hyperthermophilic archaeon, Pyrococcus furiosus. The novel endonuclease, designated as
Endonulcease Q (EndoQ), recognizes uracil, abasic site and xanthine, as well as hypoxanthine, and cuts
the phosphodiester bond at their 5′ sides. To understand the functional process involving EndoQ, we
searched for interacting partners of EndoQ and identified Proliferating Cell Nuclear Angigen (PCNA).
The EndoQ activity was clearly enhanced by addition of PCNA in vitro. The physical interaction between
the two proteins through a PIP-motif of EndoQ and the toroidal structure of PCNA are critical for the
stimulation of the endonuclease activity. These findings provide us a clue to elucidate a unique DNA
repair system in Archaea..
33. Yoshizumi Ishino, Sonoko Ishino, Takeshi YAMAGAMI, Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea. , Nucleic Acids Res. 44, 2977-2986., 10.1093/nar/gkw153, 44, 7, 2977-2986., 2016.03, The common mismatch repair system processed by
MutS and MutL and their homologs was identified
in Bacteria and Eukarya. However, no evidence of a
functional MutS/L homolog has been reported for archaeal
organisms, and it is not known whether the
mismatch repair system is conserved in Archaea.
Here, we describe an endonuclease that cleaves
double-stranded DNA containing a mismatched base
pair, from the hyperthermophilic archaeon Pyrococcus
furiosus. The corresponding gene revealed
that the activity originates from PF0012, and we
named this enzyme Endonuclease MS (EndoMS)
as the mismatch-specific Endonuclease. The sequence
similarity suggested that EndoMS is the ortholog
of NucS isolated from Pyrococcus abyssi,
published previously. Biochemical characterizations
of the EndoMS homolog from Thermococcus kodakarensis
clearly showed that EndoMS specifically
cleaves both strands of double-stranded DNA into 5′-
protruding forms, with the mismatched base pair in
the central position. EndoMS cleaves G/T, G/G, T/T,
T/C andA/Gmismatches,with amore preference for
G/T, G/G and T/T, but has very little or no effect on
C/C, A/C andA/Amismatches. The discovery of this
endonuclease suggests the existence of a novel mismatch
repair process, initiated by the double-strand
break generated by the EndoMS endonuclease, in Archaea
and some Bacteria..
34. Yoshizumi Ishino, Sonoko Ishino, Takeshi YAMAGAMI, A longer finger-subdomain of family A DNA polymerases found by metagenomic analysis strengthens DNA binding and primer extension abilities, doi: 10.1016/j.gene.2015.10.030., 576, (2 Pt 1), 690-695, 2016.02, The family A DNA polymerases from thermophilic bacteria are useful for PCR. The DNA polymerase from Thermus aquaticus (Taq polymerase) was the original enzyme used when practical PCR was developed, and it has remained the standard enzyme for PCR to date. .
35. Yoshizumi Ishino, Sonoko Ishino, makoto kimura, Mutation of the gene encoding the ribonuclease P RNA in the hyperthermophilic archaeon Thermococcus kodakarensis causes decreased growth rate and impaired processing of tRNA precursors., doi: 10.1016/j.bbrc.2015.11.012, 468, 4, 660-665, 2015.12, Ribonuclease P (RNase P) catalyzes the processing of 5' leader sequences of tRNA precursors in all three phylogenetic domains. RNase P also plays an essential role in non-tRNA biogenesis in bacterial and eukaryotic cells. For archaeal RNase Ps, additional.
36. Yoshizumi Ishino, Sonoko Ishino, Takeshi YAMAGAMI, Miyako Shiraishi, Naruto Makita, EndoQ and EndoV work individually for damaged DNA base repair in Pyrococcus furiosus., doi: 10.1016/j.biochi.2015.06.015., 118, 264-269, 2015.11, Base deamination is a typical form of DNA damage, and it must be repaired quickly to maintain the genome integrity of living organisms. Endonuclease Q (EndoQ), recently found in the hyperthermophilic archaea, is an enzyme that cleaves the phosphodiester b.
37. Yoshizumi Ishino, Sonoko Ishino, Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm., doi: 10.1093/jxb/erv325., 66, 20, 6137-6147, 2015.10, Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide ex.
38. Yoshizumi Ishino, Maiko Tanabe, Hirokazu Nishida, From structure-function analyses to protein engineering for practical applications of DNA ligase, doi: 10.1155/2015/267570, Article ID 267570, 1-20, 2015.10, DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligas.
39. Yoshizumi Ishino, Sonoko Ishino, Structural basis for substrate recognition and processive cleavage mechanisms of the trimeric exonuclease PhoExo I. , doi: 10.1093/nar/gkv654., 43, 14, 7122-7136, 2015.08, Nucleases play important roles in nucleic acid processes, such as replication, repair and recombination. Recently, we identified a novel single-strand specific 3'-5' exonuclease, PfuExo I, from the hyperthermophilic archaeon Pyrococcus furiosus, which may.
40. Yoshizumi Ishino, Functional role of the C-terminal tail of the archaeal ribosomal stalk in recruitment of two elongation factors to the sarcin/ricin loop of 23S rRNA., doi: 10.1111/gtc.12256., 43, 7, 2853-2863, 2015.06, Two types of elongation factors alternate in their binding to the factor-binding center of the ribosome. Both binding events are accompanied by GTP hydrolysis and drive the translation elongation cycle. The multicopy ribosomal protein family, termed the s.
41. Yoshizumi Ishino, Issay Narumi, DNA repair in hyperthermophilic and hyperradioresistant microorganisms., doi: 10.1016/j.mib.2015.05.010., 25, 103-112., 2015.06, The genome of a living cell is continuously under attack by exogenous and endogenous genotoxins. Especially, life at high temperature inflicts additional stress on genomic DNA, and very high rates of potentially mutagenic DNA lesions, including deaminatio.
42. Yoshizumi Ishino, Sonoko Iahino, An optimized Npro-based method for expression and purification of intrinsically disordered proteins for NMR study. ,, 3, 1, 1-6, 2015.03.
43. Yoshizumi Ishino, Sonoko Iahino, Takeshi YAMAGAMI, A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furious , doi: 10.1093/nar/gkv121, 43, 5, 2853-2863, 2015.02, DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3 from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5 from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea..
44. Yoshizumi Ishino, Sonoko Iahino, Daisuke Kohda, Disordered interdomain region of Gins is important for functional tetramer formation to stimulate MCM helicase in Thermoplasma acidophilum. ,, 79, 3, 432-438, 2014.11, The eukaryotic MCM is activated by forming the CMG complex with Cdc45 and GINS to work as a replicative helicase. The eukaryotic GINS consists of four different proteins to form tetrameric complex. In contrast, the TaGins51 protein from the thermophilic archaeon, Thermoplasma acidophilum forms a homotetramer (TaGINS), and interacts with the cognate MCM (TaMCM) to stimulate the DNA-binding, ATPase, and helicase activities of TaMCM. All Gins proteins from Archaea and Eukarya contain α-helical A- and β-stranded B-domains. Here, we found that TaGins51 forms the tetramer without the B-domain. However, the A-domain without the linker region between the A- and B-domains could not form a stable tetramer, and furthermore, the A-domain by itself could not stimulate the TaMCM activity. These results suggest that the formation of the Gins51 tetramer is necessary for MCM activation, and the disordered linker region between the two domains is critical for thefunctional complex formation..
45. Yoshizumi Ishino, Takeshi YAMAGAMI, Sonoko Iahino, Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering. , doi: 10.3389/fmicb.2014.00461, 5, 461, 1-10, 2014.09, DNApolymerasesarewidelyusedforDNAmanipulationinvitro,includingDNAcloning,sequencing,DNAlabeling,mutagenesis,andotherexperiments.ThermostableDNApolymerasesareespeciallyusefulandbecamequitevaluableafterthedevelopmentofPCRtechnology.ADNApolymerasefromThermusaquaticus(Taqpolymerase)isthemostfamousDNApolymeraseasaPCRenzyme,andhasbeenwidelyusedallovertheworld.Inthisstudy,thegenefragmentsofthefamilyADNApolymeraseswereamplifiedbyPCRfromtheDNAsfrommicroorganismswithinenvironmentalsoilsamples,usingaprimersetforthetwoconservedregions.ThecorrespondingregionofthepolgeneforTaqpolymerasewassubstitutedwiththeamplifiedgenefragments,andvariouschimericDNApolymeraseswereprepared.Basedonthepropertiesofthesechimericenzymesandtheirsequences,tworesidues,E742andA743,inTaqpolymerasewerefoundtobecriticalforitselongationability.Taqpolymeraseswithmutationsat742and743actuallyshowedhigherDNAaffinityandfasterprimerextensionability.ThesefactorsalsoaffectedthePCRperformanceoftheDNApolymerase,andimprovedPCRresultswereobservedwiththemutantTaqpolymerase.
46. Yoshizumi Ishino, Sonoko Iahino, Activation of the MCM helicase from the thermophilic archaeon, Thermoplasma acidophilum by interactions with GINS and Cdc6-2., Springer, DOI 10.1007/s00792-014-0673-6, 18, 915-924, 2014.07, In DNA replication studies, the mechanism for regulation of the various steps from initiation to elongation is a crucial subject to understand cell cycle control. The eukaryotic minichromosome maintenance (MCM) protein complex is recruited to the replication origin by Cdc6 and Cdt1 to form the pre-replication complex, and participates in forming the CMG complex formation with Cdc45 and GINS to work as the active helicase. Intriguingly, Thermoplasma acidophilum, as well as many other archaea, has only one Gins protein homolog, contrary to the heterotetramer of the eukaryotic GINS made of four different proteins. The Gins51 protein reportedly forms a homotetramer (TaGINS) and physically interacts with TaMCM. In addition, TaCdc6-2, one of the two Cdc6/Orc1 homologs in T. acidophilum reportedly stimulates the ATPase and helicase activities of TaMCM in vitro. Here, we found a reaction condition, in which TaGINS stimulated the ATPase and helicase activities of TaMCM in a concentration dependent manner. Furthermore, the stimulation of the TaMCM helicase activity by TaGINS was enhanced by the addition of TaCdc6-2. A gel retardation assay revealed that TaMCM, TaGINS, and TaCdc6-2 form a complex on
ssDNA. However, glutaraldehyde-crosslinking was necessary to detect the shifted band, indicating that the ternary complex of TaMCM–TaGINS–TaCdc6-2 is not stable in vitro. Immunoprecipitation experiment supported a weak interaction of these three proteins in vivo. Activation of the replicative helicase by a mechanism including a Cdc6-like
protein suggests the divergent evolution after the division into Archaea and Eukarya..
47. Yoshizumi Ishino, Sonoko Iahino, Takeshi YAMAGAMI, Multiple interactions of the intrinsically disordered region between the helicase and the nuclease domains of the archaeal Hef protein. , ASBMB, DOI 10.1074/jbc.M114.554998, 289, 31, 21627-21639, 2014.06, Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein.Weanalyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that
the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.
48. Yoshizumi Ishino, Sonoko Iahino, Biochemical characterization of endonuclease V from the hyperthermophilic archaeon, Pyrococcus furiosus. , Journal of Biochemistry, 10.1093/jb/mvu010. , 155, 5, 325-333, 2014.05, Endonuclease V (Endo V) is a DNA repair enzyme that recognizes deoxyinosine and cleaves the second phosphodiester bond on the 3' side of the deaminated base lesion. A database search revealed the presence of homologous genes for Endo V in most archaeal species, but the absence in some methanogenic species. We cloned a gene encoding the sequence homologous to Escherichia coli Endo V from the genome of the hyperthermophilic euryarchaeon, Pyrococcus furiosus and purified gene product (PfuEndoV) to homogeneity. In vitro characterization showed that PfuEndoV possesses specific endonuclease activity for the deoxyinosine-containing DNA strand. The activity of the enzyme was maximal at 90°C. Stable complex formation between PfuEndoV and nicked DNA produced by the cleavage reaction was detected by gel mobility shift assays. The molecular mechanisms of the inosine repair pathway including Endo V in the archaeal cells are discussed. Interestingly, PfuEndoV cleaved inosine-containing RNA strands as well as DNA substrates. PfuEndoV may also be involved in RNA metabolism. .
49. Yoshizumi Ishino, Sonoko Ishino, Mutations of Asp540 and the domain connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity., FEBS Letters, 10.1016/, 588, 230-235, 2014.01, The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity. .
50. Yoshizumi Ishino, Expression, high-pressure refolding, purification, crystallization and preliminary X-ray analysis of a novel single-strand-specific 3′–5′exonuclease PhoExo I from Pyrococcus horikoshii OT3. , doi:10.1107/S2053230X14012734, F70, 1076-1079, 2014.01, PhoExo I is a single-strand-specific 30–50 exonuclease from Pyrococcus horikoshii OT3 and is thought to be involved in a Thermococcales-specific DNA-repair pathway. The recombinant PhoExo I protein was produced as inclusion bodies in Escherichia coli cells. Solubilization of the inclusion bodies was performed by the high-pressure refolding method and highly purified protein was subjected to crystallization by the sitting-drop vapour-diffusion method at 20C. A crystal of PhoExo I was obtained in a reservoir solution consisting of 0.1 M Tris–HCl pH 8.9, 27% PEG 6000 and diffracted X-rays to 1.52 A ˚ resolution. The crystal of PhoExo I belonged to space group H32, with unit-cell parameters a = b = 112.07, c = 202.28 A ˚ . The crystal contained two PhoExo I molecules in the asymmetric unit.
51. Kazuo Tori, Sonoko Iahino, Shinichi Kiyonari, Saki Tahara, Yoshizumi Ishino, A novel single-strand specific 3'-5' exonuclease found in the hyperthermophilic archaeon,, Pyrococcus furiosus. , PLoS ONE, 10.1371/journal.pone.0058497, 8, 3, e58497-1-e58497-9, 2013.03, Nucleases play important roles in all DNA transactions, including replication, repair, and recombination. Many different nucleases from bacterial and eukaryotic organisms have been identified and functionally characterized. However, our knowledge about the nucleases from Archaea, the third domain of life, is still limited. We searched for 39–59 exonuclease activity in the hyperthermophilic archaeon, Pyrococcus furiosus, and identified a protein with the target activity. The purified protein, encoded by PF2046, is composed of 229 amino acids with a molecular weight of 25,596, and displayed singlestrand specific 39–59 exonuclease activity. The protein, designated as PfuExo I, forms a stable trimeric complex in solution and excises the DNA at every two nucleotides from the 39 to 59 direction. The amino acid sequence of this protein is conserved only in Thermococci, one of the hyperthermophilic classes in the Euryarchaeota subdomain in Archaea. The newly discovered exonuclease lacks similarity to any other proteins with known function, including hitherto reported 39–59 exonucleases. This novel nuclease may be involved in a DNA repair pathway conserved in the living organisms as a specific member for some hyperthermophilic archaea..
52. Yoshizumi Ishino, Novel inhibition of archaeal family-D DNA polymerase by uracil., Nucleic Acids Res., 41, 7, 4207-4218, 2013.02, Archaeal family-D DNA polymerase is inhibited by the presence of uracil in DNA template strands. When the enzyme encounters uracil, following three parameters change: DNA binding increases roughly 2-fold, the rate of polymerization slows by a factor of ≈ 5 and 3'-5' proof-reading exonuclease activity is stimulated by a factor of ≈ 2. Together these changes result in a significant decrease in polymerization activity and a reduction in net DNA synthesis. Pol D appears to interact with template strand uracil irrespective of its distance ahead of the replication fork. Polymerization does not stop at a defined location relative to uracil, rather a general decrease in DNA synthesis is observed. 'Trans' inhibition, the slowing of Pol D by uracil on a DNA strand not being replicated is also observed. It is proposed that Pol D is able to interact with uracil by looping out the single-stranded template, allowing simultaneous contact of both the base and the primer-template junction to give a polymerase-DNA complex with diminished extension ability..
53. Yumani Kuba, Sonoko Iahino, Takeshi YAMAGAMI, Masahiro Tokuhara, Tamotsu Kanai, Ryosuke Fujikane, Hiromi Daiyasu, Haruyuki Atomi, Yoshizumi Ishino, Comparative analyses of the two PCNAs from the hyperthermophilic archaeon, Thermococcus kodakarensis , Genes to Cells , 10.1111/gtc.12007, 17, 11, 923-937, 2012.11, The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced,
Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Dpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate
(MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain..
54. S. Ishino and Y. Ishino, Application of environmental DNA resources to create useful DNA polymerases with different properties. , Microorganisms in Sustainable Agriculture and Biotechnology. , 2012.05.
55. Yang, Y., Ishino, S., Yamagami, T., Kumamaru, T., Satoh, H., and Ishino, Y., The OsGEN-L protein from Oryza sativa possesses Holliday junction resolvase activity as well as 5′-flap endonuclease activity. , Journal of Biochemistry, 2012.05, OsGEN-L has a 5'-flap endonuclease activity and plays an essential role in rice microspore development. The Class 4 RAD2/XPG family nucleases, including OsGEN-L, were recently found to have resolving activity for the Holliday junction (HJ), the intermediate of DNA strand recombination. In this study, we performed a detailed characterization of OsGEN-L, as a structure-specific endonuclease. Highly purified OsGEN-L was prepared as the full-length protein for in vitro endonuclease assays using various structured DNAs, and the 5'-flap endonuclease activity, which is stimulated in a PCNA-dependent manner, was demonstrated. In addition, the in vitro HJ resolving activity of OsGEN-L represents the first such activity originating from plant cells. OsGEN-L cleaved HJ at symmetrically related sites of the branch point. However, the two branched strands seemed to be cleaved individually, and not cooperatively, by each OsGEN-L monomer protein. The substrate specificity suggests that OsGEN-L functions in multiple processes of DNA metabolism in rice cells..
56. Yang, Y., Ishino, S., Yamagami, T., Kumamaru, T., Satoh, H., and Ishino, Y., The OsGEN-L protein from Oryza sativa possesses Holliday junction resolvase activity as well as 5′-flap endonuclease activity. , Journal of Biochemistry, 2012.05, OsGEN-L has a 5'-flap endonuclease activity and plays an essential role in rice microspore development. The Class 4 RAD2/XPG family nucleases, including OsGEN-L, were recently found to have resolving activity for the Holliday junction (HJ), the intermediate of DNA strand recombination. In this study, we performed a detailed characterization of OsGEN-L, as a structure-specific endonuclease. Highly purified OsGEN-L was prepared as the full-length protein for in vitro endonuclease assays using various structured DNAs, and the 5'-flap endonuclease activity, which is stimulated in a PCNA-dependent manner, was demonstrated. In addition, the in vitro HJ resolving activity of OsGEN-L represents the first such activity originating from plant cells. OsGEN-L cleaved HJ at symmetrically related sites of the branch point. However, the two branched strands seemed to be cleaved individually, and not cooperatively, by each OsGEN-L monomer protein. The substrate specificity suggests that OsGEN-L functions in multiple processes of DNA metabolism in rice cells..
57. Ishino, S., Kawamura, A., and Ishino, Y., Application of PCNA to processive PCR by reducing the stability of its ring structure. , J. Jap. Soc. Extremophiles. , 11, 2, 印刷中, 2012.05.
58. Murase S, Ishino S, Ishino Y, Tanaka T. , Control of enzyme reaction by a designed metal ion-dependent α-helical coiled-coil protein. 40, J. Biol. Inorg. Chem. , 17, 5, 791-799, 2012.05, Regulation of protein function by external stimuli is a fascinating target for de novo design. We have constructed a peptide that assembles into a homotrimer in the presence of metal ions, such as Ni(2+), Cu(2+), and Zn(2+). We fused the peptide construct to the DNA-binding domain (DBD) of the heat shock factor from Saccharomyces cerevisiae, which binds tandem repeats of the heat shock element (HSE). However, the fusion protein bound to the natural three tandem HSEs even in the absence of metal ions, although mainly as the dimerized protein. Using "skipped" HSEs containing six additional nucleotides inserted between two adjacent HSEs, to prevent interactions between the DBDs, we found the fusion protein bound to the new DNA target in a metal-ion-dependent manner, as monitored by a HindIII protection assay. The fusion protein containing two metal binding sites in the metal-ion-controlled domain inhibited RNA transcription by T7 RNA polymerase in the presence of metal ions, in a template containing skipped HSEs downstream of the T7 promoter. The designed protein therefore regulates the functions of the enzyme in a metal-ion-dependent manner..
59. ISHINO Yoshizumi and ISHINO Sonoko, Rapid progress of DNA replication studies in Archaea, the third domain of life., SCIENCE CHINA  Life Sciences, doi: 10.1007/s11427-012-4324-9, 55, 1-18, 2012.05, Archaea, the third domain of life, are interesting organisms to study from the aspects of molecular and evolutionary biology. Archaeal cells have a unicellular ultrastructure without a nucleus, resembling bacterial cells, but the proteins involved in genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of Eukaryota. Therefore, archaea provide useful model systems to understand the more complex mechanisms of genetic information processing in eukaryotic cells. Moreover, the hyperthermophilic archaea provide very stable proteins, which are especially useful for the isolation of replisomal multicomplexes, to analyze their structures and functions. This review focuses on the history, current status, and future directions of archaeal DNA replication studies..
60. Kawamura, A., Ishino, Y., and Ishino, S. , Biophysical analysis of PCNA from Pyrococcus furiosus., J. Jap. Soc. Extremophiles. , 11, 2, 印刷中, 2012.02.
61. Ishino, S., Fujino, S., Tomita, H., Ogino, H., Takao, K., Daiyasu, H., Kanai, T., Atomi, H., and Ishino, Y., Biochemical and genetical analyses of the three mcm genes from the hyperthermophilic archaeon, Thermococcus kodakarensis. , Genes to Cells, 10.1111/j.1365-2443.2011.01562.x., 16, 12, 1176-1189, 2011.11, In eukaryotes, the replicative DNA helicase 'core' is the minichromosome maintenance (Mcm) complex (MCM), forming a heterohexameric complex consisting of six subunits (Mcm2-7). Recent studies showed that the CMG (Cdc45-MCM-GINS) complex is the actual helicase body in the replication fork progression complex. In Archaea, Thermococcus kodakarensis harbors three genes encoding the Mcm homologs on its genome, contrary to most archaea, which have only one homolog. It is thus, of high interest, whether and how these three Mcms share their functions in DNA metabolism in this hyperthermophile. Here, we report the biochemical properties of two of these proteins, TkoMcm1 and TkoMcm3. In addition, their physical and functional interactions with GINS, possibly an essential factor for the initiation and elongation process of DNA replication, are presented through in vitro ATPase and helicase assays, and an in vivo immunoprecipitation assay. Gene disruption and product quantification analyses suggested that TkoMcm3 is essential for cell growth and plays a key role as the main DNA helicase in DNA replication, whereas TkoMcm1 also shares some function in the cells..
62. Ogino, H., Ishino, S. Mayanagi, K., Haugland,G, H., Birkeland, N-K., Yamagishi, A., and Ishino Y., The GINS complex from the thermoacidophilic archaeon, Thermoplasma acidophilum may function as a homotetramer in DNA replication. , Extremophiles , 10.1007/s00792-011-0383-2., 15, 4, 529-539, 2011.06, The eukaryotic GINS heterotetramer, consisting of Sld5, Psf1, Psf2, and Psf3, participates in "CMG complex" formation with mini-chromosome maintenance (MCM) and Cdc45 as a key component of a replicative helicase. There are only two homologs of the GINS proteins in Archaea, and these proteins, Gins51 and Gins23, form a heterotetrameric GINS with a 2:2 molar ratio. The Pyrococcus furiosus GINS stimulates the ATPase and helicase activities of its cognate MCM, whereas the Sulfolobus solfataricus GINS does not affect those activities of its cognate MCM, although the proteins bind each other. Intriguingly, Thermoplasma acidophilum, as well as many euryarchaea, have only one gene encoding the sequence homologous to that of archaeal Gins protein (Gins51) on the genome. In this study, we investigated the biochemical properties of the gene product (TaGins51). A gel filtration and electron microscopy revealed that TaGins51 forms a homotetramer. A physical interaction between TaGins51 and TaMcm was detected by a surface plasmon resonance analysis. Unexpectedly, TaGins51 inhibited the ATPase activity, but did not affect the helicase activity of its cognate MCM. These results suggest that another factor is required to form a stable helicase complex with MCM and GINS at the replication fork in T. acidophilum cells..
63. Liu, C., McKinney, MC., Chen, YH., Earnest, TM., Shi, X., Lin, LJ., Ishino, Y., Dahmen, K., Cann, I.K., Ha, T., Reverse-Chaperoning Activity of an AAA+ Protein. , Biophysical Journal , 10.1016/j.bpj.2011.01.057, 100, 1344-1352., 2011.05.
64. Oyama T, Ishino S, Fujino S, Ogino H, Shirai T, Mayanagui K, Saito M, Nagasawa N, Ishino Y, Morikawa K., Architectures of archaeal GINS complexes, essential DNA replication initiation factors, BMC Biol., doi:10.1186/1741-7007-9-28, 9, 28, 2011.04.
65. Fujikane, R., Ishino, S., Ishino, Y., Forterre, P., Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis. , Genes Genet. Syst. , 10.1266/ggs.85.243, 85, , 243-257, 2010.08, Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins.  Accordingly, little is known about the repair pathways used by archaeal cells at high temperature.  Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis.  We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, mre11, rad50, herA, nurA, and xpg/fen1 genes.  Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to UV, methylmethane sulfonate and mitomycin C, as compared with the wild-type strain.  The hjm null mutant showed sensitivity specifically to mitomycin C.  On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage.  The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells.  Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes.  The high sensitivity of the Δhef mutants to mitomycin C suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links.  These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here..
66. Akita, M., Adachi, A, Takemura, K., Yamagami, T., Matsunaga, F., and Ishino, Y., Cdc6/Orc1 from Pyrococcus furiosus may act as the origin recognition protein and Mcm helicase recruiter. , Gens to Cells, 10.1111/j.1365-2443.2010.01402.x, 15, 5, 537-552, 2010.05.
67. Tori, K., Dassa, B., Johnson, M. A., Southworth, M. W., Brace, L. E., Ishino, Y., Pietrokovski, S., and Perler, F. B., Splicing of the Mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate Block F nucleophile., J. Biol. Chem. , 285, 4, 2515-2526, 2010.01.
68. Lin, L-J., Yoshinaga, A., Lin, Y., Guzman, C., Chen, Y-H., Mei, S., Lagunas, A. M., Koike, S., Iwai, S., Spies, M. A., Nair, S. K., Mackie, R. I., Ishino, Y., and Cann, I. K., Molecular analyses of an unusual translesion DNA polymerase from Methanosarcina acetivorans C2A., J. Mol. Biol., 397, 1, 13-30, 2010.01.
69. Nishida, H., Mayanagi, K., Kiyonari, S., Sato, Y., Oyama, T., Ishino, Y., and Morikawa, K., Structural determinant for switching between the polymerase and exonuclease modes in the PCNA-replicative DNA polymerase complex., Proc. Natl. Acad. Sci. USA. , 106, 49, 20693-20698, 2009.12.
70. Morii H, Kiyonari S, Ishino Y, and Koga Y., A novel biosynthetic pathway of archaetidyl-myo-inositol via archaetidyl-myo-inositol phosphate from CDP-archaeol and D-glucose 6-phosphate in the methanoarchaeon Methanothermobacter thermautotrophicus cells., J. Biol. Chem., 284, 45, 30766-30774, 2009.11.
71. Kiyonari, S., Tahara, S., Shirai, T., Iwai, S., Ishino, S., and Ishino, Y., Biochemical properties and BER complex formation of AP endonuclease from Pyrococcus furiosus, Nucleic Acids Res., 37, 19, 6439-6453, 2009.09.
72. Matsunaga, F., Takemura, K., Akita, M., Adachi, A., Yamagami, T., and Ishino, Y., Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus., Extremophiles, 14, 21-31, 2009.09.
73. Chen, Y-H., Lin, Y., Yoshinaga, A., Chhotani, B., Lorenzini, J., Crofts, A., Mei, S., Mackie, R.I., Ishino, Y., and Cann, I.K.O., Molecular analyses of a three-subunit euryarchaeal clamp loader complex from Methsnosarcina acetivorans. , J. Bacteriol., 191, 23, 6539-6549, 2009.08.
74. Mayanagi. K., Kiyonari, S., Saito, M., Shirai, T., Ishino, Y., and Morikawa, K., Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architechture., Proc. Natl. Acad. Sci. USA., 106, 4657-4652 , 2009.03.
75. Oyama T, Oka H, Mayanagi K, Shirai T, Matoba K, Fujikane R, Ishino Y, Morikawa K, Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm., BMC Struct. Biol., 9, 2 (1-12), 2009.03.
76. Matsukawa H, Yamagami T, Kawarabayasi Y, Miyashita Y, Takahashi M, Ishino Y., A useful strategy to construct DNA polymerases with different properties by using genetic resources from environmental DNA, Genes Genet. Syst., 84, 3-13, 2009.02.
77. S. Kiyonari, S. Tahara, M. Uchimura, T. Shirai, S. Ishino, and Yoshizumi Ishino,, Studies on the base excision repair (BER) complex in Pyrococcus furiosus., Biochem. Soc. Transact., in press, 2008.12.
78. S . Kiyonari, M. Uchimura, T. Shirai, and Yoshizumi Ishino,, Physical and Functional Interactions between Uracil-DNA glycosylase and proliferating cell nuclear antigen from the euryarchaeon Pyrococcus furiosus., J. Biol. Chem., 283, No35, 24185-24193, 2008.08.
79. Yoshimochi, T., Fujikane, R., Kawanami, M., Matsunaga, F., and Ishino, Y.*, The GINS complex from Pyrococcus furiosus stimulates the MCM helicase activity., Journal of Biological Chemistry, 283, No3, 1601-1609, 2008.01.
80. Kiyonari, S., Kamigochi, T., and Ishino, Y. , A single amino acid substitution in the DNA-binding domain of Aeropyrum pernix DNA ligase impairs its interaction with proliferating cell nuclear antigen, Extremophiles, 11, 675−684, 2007.05.
81. Tori, K., Kimizu, M., Ishino, S., and Ishino. Y. , Both DNA polymerase BI and D from the hyperthermophilic archaeon, Pyrococcus furiosus bind to PCNA at the C-terminal PIP box motifs. , Journal of Bacteriology, 189, 5652-5657, 2007.05.
82. Matsunaga, F., Glatigny, A., Mucchielli-Giorgi, M. H., Agier, N., Delacroix, H., Marisa, M., Durosay, P., Ishino, Y., Aggerbeck, L., and Forterre, P. , Genomewide and Biochemical Analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp. , Nucleic Acids Res. , 35, 3214-3222., 2007.04.
83. Nishida H, Tanabe M, Ishino Y, Oyama T, Morikawa K. , Crystallization and preliminary crystallographic study of DNA polymerase from Pyrococcus furiosus. , Protein Pept Lett. , 2007.04.
84. Imamura, K., Fukunaga, K., Kawarabayasi, Y., and Ishino, Y., Specific interactions of three PCNAs with replication-related proteins in Aeropyrum pernix., Molecular Microbiology , 64, 308-318, 2007.01.
85. Kiyonari, S., Takayama, K., Nishida, H., Ishino, Y., Identification of a novel binding motif in Pyrococcus furiosus DNA ligase for the functional interaction with proliferating cell nuclear antigen., J. Biol. Chem., 281 巻 28023-28032ページ, 2006.07.
86. Nishida, H., Matsumiya, S., Tsuchiya, D., Ishino, Y., Morikawa, K., Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus., Acta Cryst., F62巻 253-256ページ, 2006.01.
87. Nishida, H., Kiyonari, S., Ishino, Y., Morikawa, K., The closed structure of an archaeal DNA ligase from Pyrococcus furiosus., J. Mol. Biol., 360 巻 956-967ページ, 2006.01.
88. Fujikane, R., Shinagawa, H., Ishino, Y., The archaeal Hjm helicase has recQ-like functions, and may be involved in repair of stalled replication fork., Genes to Cells, 10.1111/j.1365-2443.2006.00925.x, 11, 2, 99-110, 11巻 99-110., 2005.11.
89. Miyata, T., Suzuki, H., Oyama, T., Mayanagi, K., Ishino, Y., and Morikawa, K., Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis., Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0506447102, 102, 39, 13795-13800, 102, 13795-13800, 2005.09.
90. Nishida, H., Ishino, S., Miyata, T., Morikawa, K., and Ishino, Y, Identification of the critical region in Replication factor C from Pyrococcus furiosus for the stable complex formation with proliferating cell nuclear antigen and DNA., Genes and Genetic Systems, 10.1266/ggs.80.83, 80, 2, 83-93, 80, 83-93, 2005.06.
91. Fujikane, R., Komori, K., Shinagawa, H., and Ishino, Y., Identification of a novel helicase activity unwinding branched DNAs from the hyperthermophilic archaeon, Pyrococcus furiosus., J. Biol. Chem., 10.1074/jbc.M413417200, 280, 13, 12351-12358, 280, 12351-12358., 2005.04.
92. Nishino, T., Komori, K., Ishino, Y., and Morikawa, K., Structural and functional analyses of an archaeal XPF/Rad1/Mus81 nuclease: asymmetric DNA binding and cleavage mechanisms., Structure, 10.1016/j.str.2005.04.024, 13, 8, 1183-1192, 13, 1183-1192, 2005.04.
93. Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y., and Morikawa, K., Crystal structure and functional implications of Pyrococcus furiosus Hef helicase domain involved in branched DNA processing., Structure, 10.1016/j.str.2004.11.008, 13, 1, 143-153, 13, 143-153., 2005.01.
94. Hishida, T, Han, YW, Shibata, T, Kubota, Y, Ishino, Y, Iwasaki, H, Shinagawa, H., Role of the Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks., Genes & Development, 10.1101/gad.1223804, 18, 15, 1886-1897, 18, 1886-1897, 2004.07.
95. Miyata, T, Oyama, T, Mayanagi, K, Ishino, S, Ishino, Y, Morikawa, K., The clamp-loading complex for processive DNA replication., Nature Structural and Molecular Biology, 10.1038/nsmb788, 11, 7, 632-636, 11, 632-636., 2004.06.
96. Komori K, Hidaka M, Horiuchi T, Fujikane R, Shinagawa H, Ishino Y., Cooperation of the N-terminal helicase and C-terminal endonuclease activities of archaeal Hef protein in processing stalled replication fork., Journal of Biological Chemistry, 10.1074/jbc.M409243200, 279, 51, 53175-53185, 53175-53185, 2004.01.
97. Matsumiya, S., Ishino, S., Ishino, Y., and Morikawa, K., Intermolecular ion pairs maintain toroidal structure of Pyrococcus furiosus PCNA., Prot. Sci., 12, 823-831., 2003.01.
98. Ishino, S., Oyama, T., Yuasa, M., Morikawa, K. and Ishino, Y., Mutational Analysis of Pyrococcus furiosus Replication Factor C based on the Three-Dimensional Structure., Extremophiles, 10.1007/s00792-002-0308-1, 7, 3, 169-175, 7, 169-175., 2003.01.
99. Nishino, T., Komori, K., Ishino, Y., and Morikawa, K., X-ray structural and biochemical anatomy of an archaeal XPF/Rad1/Mus81 family nuclease: a novel domain arrangement and a striking similarity between its central endonuclease domain and type II restriction enzymes., Structure, 11, 445-457., 2003.01.
100. Jolivet, E., Matsunaga, F., Ishino, Y., Forterre, P., Prieur, D., and Myllykallio, H., Physiological responses of the hyperthermophilic archaeon Pyrococus abyssi to DNA damage caused by ionizing radiation., J. Bacteiol., 10.1128/JB.185.3958-3961.2003, 185, 13, 3958-3961, 185, 3958-3961., 2003.01.
101. K. Daimon, Y. Kawarabayasi, H. Kikuchi, Y. Sako, and Y. Ishino, Three proliferating cell nuclear antigen-like proteins found in the hyperthermophilic archaeon, Aeropyrum pernix: interactions with the two DNA polymerases., J. Bacteriol., 184, 687-694., 2002.01.
102. S. Matsumiya, S. Ishino, Y. Ishino, and K. Morikawa, Physical interaction between proliferating cell nuclear antigen and replication factor C from Pyrococcus furiosus., Genes Cells,, 10.1046/j.1365-2443.2002.00572.x, 7, 9, 911-922, 7, 911-922., 2002.01.
103. K. Komori, R. Fujikane, H. Shinagawa, and Y. Ishino, Novel endonuclease in Archaea cleaving DNA with various branched structure., Genes Genet. Syst, 10.1266/ggs.77.227, 77, 4, 227-241, 77, 227-241., 2002.01.
104. Y. Ishino, and S. Ishino, Novel DNA polymerases from Euryarchaeota., Meth. Enzymol., 334, 249-260., 2001.01.
105. S. Matsumiya, Y. Ishino, and K. Morikawa, Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen of Pyrococcus furiosus., Protein Sci., 10, 17-23., 2001.01.
106. T. Nishino, K. Komori, D. Tsuchiya, Y. Ishino, and K. Morikawa, Crystal structure of the archaeal Holliday junction resolvase Hjc and implications for DNA recognition., Structure, 9, 197-204., 2001.01.
107. I. Cann, S. Ishino, M. Yuasa, H. Daiyasu, H. Toh, K. Morikawa, and Y. Ishino, Biochemical analysis of the replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus., J. Bacteriol., 183, 2614-2623., 2001.01.
108. A.A. Bocquier, L. Liu, I.K.O. Cann, D. Kohda, and Y. Ishino, Archaeal primase: bridging the gap between RNA and DNA polymerase., Curr. Biol., 11, 452-456., 2001.01.
109. K. Mayanagi, T. Miyata, T. Oyama, Y. Ishino, and K. Morikawa, Three-dimensional electron microscopy of clamp loader small subunit from Pyrococcus furiosus., J. Struct. Biol., 134, 35-45., 2001.01.
110. Y. Ishino, T. Tsurimoto, S. Ishino, and I. Cann, Functional interactions of an archaeal sliding clamp with mammalian clamp loader and DNA polymerase d., Genes Cells, 6, 699-706., 2001.01.
111. K. Komori and Y. Ishino, Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination., J. Biol. Chem., 276, 25654-25660., 2001.01.
112. T. Nishino, K. Komori, Y. Ishino, and K. Morikawa, Dissection of the regional roles of the archaeal Holliday junction resolvase Hjc by structural and mutational analyses., J. Biol. Chem., 276, 35735-35740., 2001.01.
113. H. Daiyasu, K. Osaka, Y. Ishino, and H. Toh, Expansion of the zinc metallo-hydrolase family of the b-lactamase fold., FEBS Lett., 503, 1-6., 2001.01.
114. T. Oyama, Y. Ishino, I. Cann, S. Ishino, and K. Morikawa, Atomic structure of the clamp loader small subunit from Pyrococcus furiosus., Mol. Cell, 8, 455-463., 2001.01.
115. F. Matsunaga, P. Forterre, Y. Ishino, and H. Myllykallio, In vivo interactions of archaeal Cdc6/Orcl and minichromosome maintenance proteins with the replication origin., Proc. Natl. Acad. Sci.USA, 98, 11152-11157., 2001.01.
116. L. Liu, K. Komori, S. Ishino, A. Bocquier, I. Cann, D. Kohda, and Y. Ishino, The archaeal DNA primase: biochemical characterization of the p41-p46 complex from Pyrococcus furiosus., J. Biol. Chem., 276, 45484-45490., 2001.01.
117. K. Komori and Y. Ishino, Functional interdependence of DNA polymerizing and 3'→5' exonucleolytic activities in Pyrococcus furiosus DNA polymerase I., Protein Engineering, 13, 41-47., 2000.01.
118. K. Ichiyanagi, Y. Ishino, M. Ariyoshi, K. Komori, and K. Morikawa, Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI., J. Mol. Biol., 300, 889-901., 2000.01.
119. K. Komori, T. Miyata, J. DiRuggiero, R. Holley-Shanks, I. Hayashi, I. Cann, K. Mayanagi, H. Shinagawa, and Y. Ishino, Both RadA and RadB are involved in homologous recombination in Pyrococcus furiosus., J. Biol. Chem., 275, 33782-33790., 2000.01.
120. K. Komori, T. Miyata, H. Daiyasu, H. Toh, H. Shinagawa, and Y. Ishino, Domain analysis of an archaeal RadA protein for the strand exchange activity., J. Biol. Chem., 275, 33791-33797., 2000.01.
121. H. Daiyasu, K. Komori, S. Sakae, Y. Ishino, and H. Toh, Hjc resolvase is a distantly related member of the Type II restriction endonuclease family., Nucleic Acids Res., 28, 4540-4543., 2000.01.
122. K. Komori, S. Sakae, R. Fujikane, K. Morikawa, H. Shinagawa, and Y. Ishino, Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus., Nucleic Acids Res., 28, 4544-4551., 2000.01.
123. K. Komori, S. Sakae, H. Daiyasu, H. Toh, K. Morikawa, H. Shinagawa, and Y. Ishino, Mutational analysis of the Pyrococcus furiosus Holliday junction resolvase Hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities., J. Biol. Chem., 275, 40385-40391., 2000.01.
124. T. Ohnishi, H. Iwasaki, Y. Ishino, S. Kuramitsu, A. Nakata, and H. Shinagawa, dentification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions., Genes Genet. Syst., 75, 233-243., 2000.01.
125. K. Komori, S. Sakae, H. Shinagawa, K. Morikawa, and Y. Ishino, A novel Holliday junction resolvase: functional similarity to E. coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea., Proc. Natl. Acad. Sci.USA, 96, 8873-8878., 1999.01.
126. I. Cann, S. Ishino, N. Nomura, Y. Sako, and Y. Ishino, Two family B DNA polymerases in Aeropyrum pernix, an obligate aerovic hyperthermophilic crenarchaeote., J. Bacteriol., 181, 5984-5992., 1999.01.
127. I. Cann, S. Ishino, I. Hayashi, K. Komori, H. Toh, K. Morikawa and Y. Ishino, Functional interactions of a homolog of proliferating cell nuclear antigen with DNA polymerases in Archaea., J. Bacteriol., 181, 6591-6599., 1999.01.
128. K. Komori, N. Fujita, K. Ichiyanagi, H. Shinagawa, K. Morikawa, and Y. Ishino, PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-pype endonuclease activities., Nucleic Acids Res., 27, 4167-4174., 1999.01.
129. K. Komori, K. Ichiyanagi, K. Morikawa, and Y. Ishino, PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization of the binding and cleavage abilities by site-directed mutagenesis., Nucleic Acids Res., 27, 41 75-4182., 1999.01.
130. I. Hayashi, K. Morikawa, and Y. Ishino, Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmcl homolog, in Pyrococcus furiosus., Nucleic Acids Res., 27, 4695-4702., 1999.01.
131. I. Cann and Y. Ishino, A tRNAGlu gene from the hyperthermophilic archaeon Pyrococcus furiosus contains the 3'-terminal CCA sequence of the mature tRNA., FEMS Microbiol. Lett., 160, 199-204., 1998.01.
132. I. Cann, S. Kanai, H. Toh, and Y. Ishino, Adenylosuccinate synthetase genes: molecular cloning and phylogenetic analysis of a highly conserved archaeal gene., System. Appl. Microbiol., 21, 478-486., 1998.01.
133. Y. Ishino, K. Komori, I. Cann, and Y. Koga, A novel DNA polymerase family found in Archaea., J. Bacteriol., 180, 2232-2236., 1998.01.
134. I. Cann, K. Komori, H. Toh, S. Kanai, and Y. Ishino, A heterodimeric DNA polymerase: evidence that members of the euryarchaeota possess a novel DNA polymerase., Proc. Natl. Acad. Sci.USA, 95, 14250-14255., 1998.01.
135. Yoshizumi Ishino, Sonoko Iahino, A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm., Plant Physiol. , 162, 2, 663-674, Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, β-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds..