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Yoshizumi Ishino - One of the best experts on this subject based on the ideXlab platform.
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genomewide and biochemical analyses of DNA binding activity of cdc6 orc1 and mcm proteins in pyrococcus sp
Nucleic Acids Research, 2007Co-Authors: Fujihiko Matsunaga, Yoshizumi Ishino, Annie Glatigny, Marieha La Ne Mucchielligiorgi, Nicolas Agier, Herva Delacroix, Laetitia Marisa, Patrice Durosay, Lawrence P Aggerbeck, Patrick ForterreAbstract:The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA Polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.
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biochemical analysis of replication factor c from the hyperthermophilic archaeon pyrococcus furiosus
Journal of Bacteriology, 2001Co-Authors: Isaac K. O. Cann, Sonoko Ishino, Mihoko Yuasa, Hiromi Daiyasu, Yoshizumi IshinoAbstract:In the eukaryotic DNA replication system, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are DNA Polymerase auxiliary proteins implicated in replicative and repair DNA synthesis (reviewed in reference 42). The replicative DNA Polymerases (polδ and polɛ) in Eucarya are highly processive, i.e., they can polymerize long stretches of DNA without dissociating from the template. This property is conferred upon both DNA Polymerases by PCNA, a ring-shaped homotrimeric protein capable of encircling and sliding along duplex DNA. PCNA works as an elongation factor for DNA Polymerases by tethering the Polymerases to the DNA template. For the loading of PCNA onto DNA, a clamp loader consisting of four distinct small subunits and one large subunit is required. The clamp loader, commonly known as RFC, performs this function in an ATP-dependent manner by (i) recognizing the primer terminus, (II) binding to and opening the donut-shaped PCNA, and (IIi) linking the opened PCNA topologically to the DNA. In the bacteria and bacteriophage systems, the replicative DNA Polymerases also require the clamp molecule for their processive DNA synthesis. The molecular mechanisms of the clamp-loading process have been basically conserved, although the amino acid sequences of each molecule are distinctly different from those of eukaryotic proteins. Escherichia coli DNA Polymerase III (Pol III) γ-subunit and T4 gp44/gp62 are well known as the clamp loaders for their sliding clamps, Pol III β-subunit and T4 gp45, respectively (20, 44). Since the discovery of Archaea, the third domain of life, the molecular mechanisms of their DNA transactions have become a very interesting subject. However, the current knowledge of the archaeal DNA replication mechanism is still rudimentary. Moreover, an understanding of how the hyperthermophilic Archaea maintain their genetic information systems in cells growing under conditions unfavorable to the stability of DNA is of particular interest to biologists. Several genes encoding eukaryotic-like DNA replication proteins are present in archaeal genomes (4, 7, 12, 24). This has led to the proposal that the archaeal DNA replication mechanism is basically similar to that of Eucarya. Except for the euryarchaeotic heterodimeric DNA Polymerase (3, 11, 13, 40), all archaeal DNA Polymerases described to date are single subunit proteins with sequences similar to those of the family B (α-like) DNA Polymerases, which include the chromosomal DNA replicases of Eucarya (4, 12, 32). The archaeal family B DNA Polymerases have low processivity in vitro, and their ability to replicate the genome has been questioned (29). Our recent results, however, show that the rates of DNA synthesis by Pyrococcus furiosus DNA Polymerase I (Pol BI) and DNA Polymerase II (Pol D) are enhanced by the addition of P. furiosus PCNA (PfuPCNA) (5). Surprisingly, we found that PfuPCNA can self-assemble onto circular DNA without the assistance of RFC in vitro, even though the genomes of Archaea, including the pyrococci, contain genes encoding RFC-like proteins (4). Recent reports have shown that the two-subunit RFCs from Methanobacterium thermoautotrophicum and Sulfolobus solfataricus function to load the PCNA homologs in these organisms onto the DNA strand (21, 33). To determine the functions of the two RFC-like proteins in P. furiosus, the corresponding genes located in tandem in the genome were cloned separately and expressed in E. coli, and their products were biochemically characterized in this study. The results of our analyses provide evidence for the basic conservation of the role of RFC in DNA synthesis in both Archaea and Eucarya.
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a heterodimeric DNA Polymerase evidence that members of euryarchaeota possess a distinct DNA Polymerase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Isaac K. O. Cann, Kayoko Komori, Satoru Kanai, Yoshizumi IshinoAbstract:We describe here a DNA Polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA Polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA Polymerase δ, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA Polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA Polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-Polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA Polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.
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a heterodimeric DNA Polymerase evidence that members of euryarchaeota possess a distinct DNA Polymerase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Isaac K. O. Cann, Kayoko Komori, Satoru Kanai, Yoshizumi IshinoAbstract:We describe here a DNA Polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA Polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA Polymerase δ, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA Polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA Polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-Polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA Polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.
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organization and nucleotide sequence of the DNA Polymerase gene from the archaeon pyrococcus furiosus
Nucleic Acids Research, 1993Co-Authors: Takashi Uemori, Yoshizumi Ishino, Hiroyuki Toh, Kiyozo Asada, Ikunoshin KatoAbstract:We cloned the gene encoding the thermostable DNA Polymerase from the archaeon Pyrococcus furiosus. The DNA fragment of 2785 base pair (bp) containing the structural gene for DNA Polymerase was sequenced. DNA Polymerase (Pfu Polymerase), as deduced from the DNA sequence, consisted of 775 amino acids, had a molecular weight of 90, 109, and was structurally homologous to the alpha-like DNA Polymerases (family B) represented by human DNA Polymerase alpha and Escherichia coli DNA Polymerase II. An unrooted phylogenetic tree of the alpha-like DNA Polymerases based on the amino acid sequence alignment was constructed. Pfu Polymerase, with two other archaeon Polymerases, constitutes a group with some animal viruses. The transcription initiation sites of the pol gene were identified by analysis of in vivo transcripts of both from P. furiosus and E. coli, and the promoters were assigned upstream of the pol coding region. A typical promoter sequence for the archaeon was found at a reasonable distance from the transcription initiation site in P. furiosus.
Akio Sugino - One of the best experts on this subject based on the ideXlab platform.
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DNA Polymerase epsilon encoded by cdc20 is required for chromosomal DNA replication in the fission yeast schizosaccharomyces pombe
Genes to Cells, 1998Co-Authors: Akio Sugino, Takeshi Ohara, Josef Sebastian, Naomi Nakashima, Hiroyuki ArakiAbstract:Background DNA Polymerase II (PolII), the homologue of mammalian DNA Polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA Polymerase epsilon. It has been shown that DNA Polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. Results We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA Polymerase epsilon (Pol epsilon). The predicted amino acid sequence of Pol epsilon is highly homologous to that of S. cerevisiae PolII and human Pol epsilon. Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. Conclusion These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.
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dpb11 which interacts with DNA Polymerase II epsilon in saccharomyces cerevisiae has a dual role in s phase progression and at a cell cycle checkpoint
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Hiroyuki Araki, Sun-hee Leem, Amornrat Phongdara, Akio SuginoAbstract:Abstract DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA Polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA Polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.
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DNA Polymerase II, the Epsilon Polymerase of Saccharomyces cerevisiae
Progress in nucleic acid research and molecular biology, 1993Co-Authors: Alan Morrison, Akio SuginoAbstract:Publisher Summary This chapter discusses the data on DNA Polymerase II in the context of current knowledge of eukaryotic DNA Polymerases, DNA replication and its fidelity, and DNA repair. DNA Polymerase II has been proffered as the “repair Polymerase.” Contrary to the implicit assumption, DNA repair is not a single process but an array of different pathways that have been dissected at least partially by genetic studies. It cannot be presumed that a particular Polymerase acts in repair but not in replication, or that a single Polymerase performs all DNA repair, or that each repair pathway has a dedicated Polymerase. If DNA Polymerase II is involved in the repair of damaged DNA, it might be possible in principle to obtain radiation-sensitive mutants either in POL2 , or in DPB2, or DPB3. Further evidence implicating DNA Polymerase II in DNA replication comes from the spontaneous mutator phenotype of the exonuclease-deficient pol2-4 mutant. While a spontaneous mutator phenotype may also arise in DNA repair mutants, the pol2-4 mutation does not confer sensitivity to DNA-damaging agents, and the epistatic relationships discussed in the chapter, link the DNA Polymerase II 3’→5’ exonuclease with DNA replication.
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DNA Polymerase II the probable homolog of mammalian DNA Polymerase epsilon replicates chromosomal DNA in the yeast saccharomyces cerevisiae
The EMBO Journal, 1992Co-Authors: Hiroyuki Araki, P Ropp, A L Johnson, L H Johnston, Alan Morrison, Akio SuginoAbstract:Abstract Two temperature-sensitive DNA Polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA Polymerase II activity partially purified from both mutants was thermolabile, while DNA Polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA Polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA Polymerases of yeast and mammals make it likely that mammalian DNA Polymerase epsilon too is required for chromosomal DNA replication.
Hiroyuki Araki - One of the best experts on this subject based on the ideXlab platform.
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DNA Polymerase epsilon encoded by cdc20 is required for chromosomal DNA replication in the fission yeast schizosaccharomyces pombe
Genes to Cells, 1998Co-Authors: Akio Sugino, Takeshi Ohara, Josef Sebastian, Naomi Nakashima, Hiroyuki ArakiAbstract:Background DNA Polymerase II (PolII), the homologue of mammalian DNA Polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA Polymerase epsilon. It has been shown that DNA Polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. Results We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA Polymerase epsilon (Pol epsilon). The predicted amino acid sequence of Pol epsilon is highly homologous to that of S. cerevisiae PolII and human Pol epsilon. Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. Conclusion These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.
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dpb11 which interacts with DNA Polymerase II epsilon in saccharomyces cerevisiae has a dual role in s phase progression and at a cell cycle checkpoint
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Hiroyuki Araki, Sun-hee Leem, Amornrat Phongdara, Akio SuginoAbstract:Abstract DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA Polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA Polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.
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DNA Polymerase II the probable homolog of mammalian DNA Polymerase epsilon replicates chromosomal DNA in the yeast saccharomyces cerevisiae
The EMBO Journal, 1992Co-Authors: Hiroyuki Araki, P Ropp, A L Johnson, L H Johnston, Alan Morrison, Akio SuginoAbstract:Abstract Two temperature-sensitive DNA Polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA Polymerase II activity partially purified from both mutants was thermolabile, while DNA Polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA Polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA Polymerases of yeast and mammals make it likely that mammalian DNA Polymerase epsilon too is required for chromosomal DNA replication.
Isaac K. O. Cann - One of the best experts on this subject based on the ideXlab platform.
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biochemical analysis of replication factor c from the hyperthermophilic archaeon pyrococcus furiosus
Journal of Bacteriology, 2001Co-Authors: Isaac K. O. Cann, Sonoko Ishino, Mihoko Yuasa, Hiromi Daiyasu, Yoshizumi IshinoAbstract:In the eukaryotic DNA replication system, replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are DNA Polymerase auxiliary proteins implicated in replicative and repair DNA synthesis (reviewed in reference 42). The replicative DNA Polymerases (polδ and polɛ) in Eucarya are highly processive, i.e., they can polymerize long stretches of DNA without dissociating from the template. This property is conferred upon both DNA Polymerases by PCNA, a ring-shaped homotrimeric protein capable of encircling and sliding along duplex DNA. PCNA works as an elongation factor for DNA Polymerases by tethering the Polymerases to the DNA template. For the loading of PCNA onto DNA, a clamp loader consisting of four distinct small subunits and one large subunit is required. The clamp loader, commonly known as RFC, performs this function in an ATP-dependent manner by (i) recognizing the primer terminus, (II) binding to and opening the donut-shaped PCNA, and (IIi) linking the opened PCNA topologically to the DNA. In the bacteria and bacteriophage systems, the replicative DNA Polymerases also require the clamp molecule for their processive DNA synthesis. The molecular mechanisms of the clamp-loading process have been basically conserved, although the amino acid sequences of each molecule are distinctly different from those of eukaryotic proteins. Escherichia coli DNA Polymerase III (Pol III) γ-subunit and T4 gp44/gp62 are well known as the clamp loaders for their sliding clamps, Pol III β-subunit and T4 gp45, respectively (20, 44). Since the discovery of Archaea, the third domain of life, the molecular mechanisms of their DNA transactions have become a very interesting subject. However, the current knowledge of the archaeal DNA replication mechanism is still rudimentary. Moreover, an understanding of how the hyperthermophilic Archaea maintain their genetic information systems in cells growing under conditions unfavorable to the stability of DNA is of particular interest to biologists. Several genes encoding eukaryotic-like DNA replication proteins are present in archaeal genomes (4, 7, 12, 24). This has led to the proposal that the archaeal DNA replication mechanism is basically similar to that of Eucarya. Except for the euryarchaeotic heterodimeric DNA Polymerase (3, 11, 13, 40), all archaeal DNA Polymerases described to date are single subunit proteins with sequences similar to those of the family B (α-like) DNA Polymerases, which include the chromosomal DNA replicases of Eucarya (4, 12, 32). The archaeal family B DNA Polymerases have low processivity in vitro, and their ability to replicate the genome has been questioned (29). Our recent results, however, show that the rates of DNA synthesis by Pyrococcus furiosus DNA Polymerase I (Pol BI) and DNA Polymerase II (Pol D) are enhanced by the addition of P. furiosus PCNA (PfuPCNA) (5). Surprisingly, we found that PfuPCNA can self-assemble onto circular DNA without the assistance of RFC in vitro, even though the genomes of Archaea, including the pyrococci, contain genes encoding RFC-like proteins (4). Recent reports have shown that the two-subunit RFCs from Methanobacterium thermoautotrophicum and Sulfolobus solfataricus function to load the PCNA homologs in these organisms onto the DNA strand (21, 33). To determine the functions of the two RFC-like proteins in P. furiosus, the corresponding genes located in tandem in the genome were cloned separately and expressed in E. coli, and their products were biochemically characterized in this study. The results of our analyses provide evidence for the basic conservation of the role of RFC in DNA synthesis in both Archaea and Eucarya.
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a heterodimeric DNA Polymerase evidence that members of euryarchaeota possess a distinct DNA Polymerase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Isaac K. O. Cann, Kayoko Komori, Satoru Kanai, Yoshizumi IshinoAbstract:We describe here a DNA Polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA Polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA Polymerase δ, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA Polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA Polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-Polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA Polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.
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a heterodimeric DNA Polymerase evidence that members of euryarchaeota possess a distinct DNA Polymerase
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Isaac K. O. Cann, Kayoko Komori, Satoru Kanai, Yoshizumi IshinoAbstract:We describe here a DNA Polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA Polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA Polymerase δ, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA Polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA Polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-Polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA Polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.
Ikunoshin Kato - One of the best experts on this subject based on the ideXlab platform.
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organization and nucleotide sequence of the DNA Polymerase gene from the archaeon pyrococcus furiosus
Nucleic Acids Research, 1993Co-Authors: Takashi Uemori, Yoshizumi Ishino, Hiroyuki Toh, Kiyozo Asada, Ikunoshin KatoAbstract:We cloned the gene encoding the thermostable DNA Polymerase from the archaeon Pyrococcus furiosus. The DNA fragment of 2785 base pair (bp) containing the structural gene for DNA Polymerase was sequenced. DNA Polymerase (Pfu Polymerase), as deduced from the DNA sequence, consisted of 775 amino acids, had a molecular weight of 90, 109, and was structurally homologous to the alpha-like DNA Polymerases (family B) represented by human DNA Polymerase alpha and Escherichia coli DNA Polymerase II. An unrooted phylogenetic tree of the alpha-like DNA Polymerases based on the amino acid sequence alignment was constructed. Pfu Polymerase, with two other archaeon Polymerases, constitutes a group with some animal viruses. The transcription initiation sites of the pol gene were identified by analysis of in vivo transcripts of both from P. furiosus and E. coli, and the promoters were assigned upstream of the pol coding region. A typical promoter sequence for the archaeon was found at a reasonable distance from the transcription initiation site in P. furiosus.