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Yoshizumi Ishino Last modified date:2022.07.25

Graduate School
Undergraduate School
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Academic Degree
Doctor of Phamaceutical Science
Country of degree conferring institution (Overseas)
Field of Specialization
Molecular Biology, Genetic Engineering, Protein Engineering
Total Priod of education and research career in the foreign country
Research Interests
  • Monitoring of mrine eovironment by metagenomic analysis
    keyword : metagenome, NGS, DNA sequencing, marine
  • Target recognition and expression mechanism of intrinsically disordered protein

    keyword : intrinsically disordered protein, molecular recognition, protein structure
  • Molecular Mechanism of DNA replication
    keyword : DNA replication, molecular mechanism, archaea, hyperthermophiles
  • Molecular mechanisms of DNA recombinatinal repair and genome integrity
    keyword : DNA repair, DNA recombination, endonucelase, homologous recombination, Holliday junction
  • Application of the enzymes and proteins to the development of novel techniques for genetic engineering
    keyword : Genetic Engineering, Gene amplification, Recombinant DNA, NUcleic Acid-relarted enzyme
Current and Past Project
  • Target recognition and expression mechanism of intrinsically disordered protein
Academic Activities
1. Yoshizumi Ishino, Sonoko Iahino, DNA replication in thermophilic microorganisms, In Thermophilic Microorganisms. Horizon Scientific Press, Norwich, UK, Horizon Scientific Press, Norwich, UK, 2015.05, DNA replication is essential for maintaining genetic information and transferring it from ancestor to descendant. To protect the genetic information from mutations, biological organisms have acquired several types of DNA repair systems. The extreme thermophiles living on the earth are microorganisms from the Bacteria and Archaea domains. The structures of the bacterial and archaeal genomes are circular, and the mechanism of replication initiation, by the binding of the initiator protein to the replication origin (oriC), is conserved in the two domains. The elongation process is also conserved, because similar primase, helicase, polymerase, and ligase functions are observed in the two domains. However, the proteins involved in the DNA replication process are quite different between Bacteria and Archaea, and their replication machineries seem to have evolved independently. The DNA repair system in extreme thermophiles should work efficiently to maintain their genome integrity at high temperatures. However, the DNA repair systems are diverse and still not comprehensively understood yet, although research in this field has been actively pursued in the systems from Escherichia coli to human. In this chapter, we focus on DNA replication in the thermophilic bacteria and archaea, and summarize the current understanding of the molecular mechanisms of replication in thermophilic microorganisms.

2. Yoshizumi Ishino, Sonoko Iahino, The Mechanisms of DNA Replication
DNA Replication in Archaea, the Third Domain of Life
, INTECH d.o.o., Rijeka, Croatia, pp. 91-126, 2013.03.
3. Sonoko Iahino, Yoshizumi Ishino, Thermophilic microbes in environmental and industrial biotechnology
DNA polymerases and DNA ligases
, Springer, 429-457, 2013.03.
4. Sonoko Iahino, Yoshizumi Ishino, Microorganisms in Sustainable Agriculture and Biotechnology.
Application of environmental DNA resources to create useful DNA polymerases with different properties.
, Springer, pp. 663-678, 2012.03.
1. Ishino, Y., Krupovic, M., Forterre, P. , History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology. , Journal of Bacteriology, doi: 10.1128/JB.00580-17., 2018.03, 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.

2. Yoshizumi Ishino, Sonoko Iahino, DNA polymerases as useful reagents for biotechnology - the history of developmental research in the field. , 2014.08, DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands according to the template DNA in the living cells. Multiple enzymes have been identified from each organism, and the shared functions of these enzymes have been investigated. In addition to their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labeling, mutagenesis, and other experiments. The fundamental ability of DNA polymerases to synthesize a deoxyribonucleotide chain is conserved. However, the more specific properties, including processivity, fidelity (synthesis accuracy), and substrate nucleotide selectivity, differ among the enzymes. The distinctive properties of each DNA polymerase may lead to the potential development of unique reagents, and therefore searching for novel DNA polymerase has been one of the major focuses in this research field. In addition, protein engineering techniques to create mutant or artificial DNA polymerases have been successfully developing powerful DNA polymerases, suitable for specific purposes among the many kinds of DNA manipulations. Thermostable DNA polymerases are especially important for PCR-related techniques in molecular biology. In this review, we summarize the history of the research on developing thermostable DNA polymerases as reagents for genetic manipulation and discuss the future of this research field..
3. Yoshizumi Ishino, Sonoko Iahino, The archaeal DNA replication machinery: past, present and future. , Genes and Genetic Systems, 2013.12, Living organisms are divided into three domains: Bacteria, Archaea, and Eukarya. Whereas Bacteria and Archaea are both prokaryotes, proteins involved in information processes; replication, transcription, and translation, are more similar in Archaea and Eukarya. Here the history of the research on archaeal DNA replication is summarized and the future of the field is discussed..
4. Ishino, Y. and Ishino, S., Rapid progress of DNA replication studies in Archaea, the third domain of life. , Sci. China, Ser-C Life Sci. , 2012.05, Archaea, the third domain of life, is a very interesting living organism to study from the aspects of molecular and evolutional biology. Archaeal cells have a unicellular ultrastructure without a nucleus, resembling bacterial cells, but the, proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of Eukaryota. Therefore, the archaeal processes provide useful model systems to understand the more complex mechanisms of genetic information processing in the 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..
5. Ishino, S and Ishino, Y., Application of environmental DNA resources to create useful DNA polymerases with different properties., Microorganisms in Sustainable Agriculture and Biotechnology. (Springer Science+Business Media), 2012.01, DNA polymerases use deoxynucleotide triphosphates to synthesize new DNA strands according to the template DNA during DNA replication and repair, and are essential to maintain genome integrity in DNA metabolism. In addition, these enzymes are widely used for genetic engineering techniques, including dideoxy-sequencing, PCR, DNA labeling, mutagenesis, and other in vitro gene manipulations. Thermostable DNA polymerases are especially useful for PCR and cycle-sequencing. We describe in this chapter a powerful strategy to use environmental DNA as a genetic resource to create useful DNA polymerases. The region corresponding to the active center of the DNA polymerizing reaction in the structural gene of well known DNA polymerases, such as Pfu DNA polymerase and Taq DNA polymerase, can be substituted with gene fragments amplified by PCR from DNAs within soil samples from various world-wide locations. The constructed chimeric pol genes can be expressed in E. coli, and the produced chimeric enzymes, possessing DNA polymerase activities with different properties, can be evaluated in terms of their processivity, fidelity, and efficiency of primer usage, to select valuable DNA polymerases for genetic engineering techniques..
1. Suzuki, S., Kurosawa, N., Yamagami, T., Matsumoto, S., Numata, T.,Ishino, S., and Ishino, Y., Genetic and Biochemical Characterizations of aLhr1 Helicase in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius., Catalysts, doi: 10.3390/catal12010034, 12, 34, 1-20, 2022.02.
2. Oki, K., Yamagami, T., Nagata, M., Mayanagi, K., Shirai T., Adachi, N., Numata, T.,Ishino, S., and Ishino, Y., DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen, Nucleic Acids Res. , 10.1093/nar/gkab243, 49, 8, 4599-4612, 2021.05.
3. Oki, K., Nagata, M., Yamagami, T.,Numata, T., @Ishino, S., Oyama, O., and @Ishino, Y., Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome, Nucleic Acids Res. , 10.1093/nar/gkab799, 50, 7, 3601-3615, 2022.04.
4. 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.
5. 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.
6. 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..
7. 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..
8. 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..
9. 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..
10. 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..
11. 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..
12. 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..
13. 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..
14. 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..
15. 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.

16. 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..
17. 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.".
18. 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
19. 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..
20. 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..
21. 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..
22. 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..
23. 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..
24. 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..
25. 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..
26. 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..
27. 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. .
28. 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.
29. 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.
30. 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..
31. 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..
32. 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.
33. 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..
34. 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..
35. 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.
36. 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..
37. 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..
38. 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.
39. 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..
40. 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.
41. 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.
42. 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.
43. 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.
44. 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.
45. 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.
46. 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.
47. 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.
48. 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.
49. 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.
50. 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.
51. 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.
52. 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.
53. 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.
54. 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.
55. 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.
1. Ishino Y. (2008) Contribution of the proteins from hyperthermophilic archaea to structural and functional analyses of DNA replication/repair apparatus. Keynote Presentation at Extremophiles 2008 Sept. 7-11, Cape Town, South Africa..
  • Genetics Promotion Award by Genetics Promotion foundation