Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Sonoko Ishino Last modified date:2019.06.24

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


Papers
1. 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..
2. 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..
3. 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..
4. 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..
5. 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..
6. 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..
7. 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..
8. Mariko Nagata, Sonoko Ishino, Takeshi Yamagami, Jan Robert Simons, Tamotsu Kanai, Haruyuki Atomi, Yoshizumi Ishino, Possible function of the second RecJ-like protein in stalled replication fork repair by interacting with Hef, Scientific reports, 10.1038/s41598-017-17306-0, 7, 1, 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..
9. Mariko Nagata, Sonoko Ishino, Takeshi Yamagami, Hiromi Ogino, Jan Robert Simons, Tamotsu Kanai, Haruyuki Atomi, Yoshizumi Ishino, The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis, Nucleic acids research, 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 GANGINS 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 Agan mutant did not grow at 93 C. Furthermore, the Agan 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..
10. Ying Liu, Sonoko Ishino, Yoshizumi Ishino, Gérard Pehau-Arnaudet, Mart Krupovic, David Prangishvili, A novel type of polyhedral viruses infecting hyperthermophilic archaea, Journal of Virology, 10.1128/JVI.00589-17, 91, 13, 2017.07, 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..
11. Hiromi Ogino, Sonoko Ishino, Daisuke Kohda, Yoshizumi Ishino, The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3′-5′ exonuclease that associates with a DNA replication complex, Journal of Biological Chemistry, 10.1074/jbc.M116.767921, 292, 19, 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..
12. Takuya Yoda, Maiko Tanabe, Toshiyuki Tsuji, Takao Yoda, Sonoko Ishino, Tsuyoshi Shirai, Yoshizumi Ishino, Haruko Takeyama, Hirokazu Nishida, Exonuclease processivity of archaeal replicative DNA polymerase in association with PCNA is expedited by mismatches in DNA, Scientific reports, 10.1038/srep44582, 7, 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..
13. Miyako Shiraishi, Sonoko Ishino, Isaac Cann, Yoshizumi Ishino, A functional endonuclease Q exists in the bacterial domain
Identification and characterization of endonuclease Q from Bacillus pumilus, Bioscience, Biotechnology and Biochemistry, 10.1080/09168451.2016.1277946, 81, 5, 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..
14. Takuji Oyama, Sonoko Ishino, Tsuyoshi Shirai, Takeshi Yamagami, Mariko Nagata, Hiromi Ogino, Masami Kusunoki, Yoshizumi Ishino, 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 research, 10.1093/nar/gkw789, 44, 19, 9505-9517, 2016.11, 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, from a 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..
15. Setsu Nakae, Atsushi Hijikata, Toshiyuki Tsuji, Kouki Yonezawa, Ken ichi Kouyama, Kouta Mayanagi, Sonoko Ishino, Yoshizumi Ishino, Tsuyoshi Shirai, Structure of the EndoMS-DNA Complex as Mismatch Restriction Endonuclease, Structure, 10.1016/j.str.2016.09.005, 24, 11, 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 double-stranded 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..
16. Miyako Shiraishi, Sonoko Ishino, Kotaro Yoshida, Takeshi Yamagami, Isaac Cann, Yoshizumi Ishino, PCNA is involved in the EndoQ-mediated DNA repair process in Thermococcales, Scientific reports, 10.1038/srep25532, 6, 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..
17. Sonoko Ishino, Yuki Nishi, Soichiro Oda, Takashi Uemori, Takehiro Sagara, Nariaki Takatsu, Takeshi Yamagami, Tsuyoshi Shirai, Yoshizumi Ishino, Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea, Nucleic acids research, 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 and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. 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..
18. Takeshi Yamagami, Hiroaki Matsukawa, Sae Tsunekawa, Yutaka Kawarabayasi, Sonoko Ishino, Yoshizumi Ishino, A longer finger-subdomain of family A DNA polymerases found by metagenomic analysis strengthens DNA binding and primer extension abilities, Gene, 10.1016/j.gene.2015.10.030, 576, 2, 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. Knowledge gained from structure-function relationship studies of Taq polymerase is applicable to create PCR enzymes with enhanced performance. We collected the deduced amino acid sequences of the regions from motif A to motif C in the family A DNA polymerases from metagenomic sequence data, obtained by sequencing DNAs from microorganisms isolated from various hot spring areas in Japan. The corresponding regions of the polA gene for Taq polymerase were substituted with the metagenomic DNA gene fragments, and various chimeric DNA polymerases were prepared. Based on the properties of these chimeric enzymes and their sequences, we found an insertion sequence that affects the primer extension ability of the family A DNA polymerases. The insertion sequence is located in the finger subdomain, and it may enhance the affinity of the enzyme to DNA. Mutant Taq polymerases with the corresponding 9 amino acid insertion displayed enhanced PCR performance..
19. Javier Abellón-Ruiz, Sonoko Ishino, Yoshizumi Ishino, Bernard A. Connolly, 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, 10.1155/2016/1510938, 2016, 2016.01, 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 primer-templates 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 DNA mutations..
20. Javier Abellón-Ruiz, Sonoko Ishino, Yoshizumi Ishino, Bernard A. Connolly, 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, 10.1155/2016/1510938, 2016, 2016.01, 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 primer-templates 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 DNA mutations..