The Experts below are selected from a list of 171 Experts worldwide ranked by ideXlab platform
Yeon Soo Seo - One of the best experts on this subject based on the ideXlab platform.
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characterization of the enzymatic properties of the yeast dna2 helicase endonuclease suggests a new model for okazaki fragment processing
Journal of Biological Chemistry, 2000Co-Authors: Sung-ho Bae, Yeon Soo SeoAbstract:Abstract The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki fragment maturation during lagging strand synthesis. In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes. We found that the Dna2 helicase activity translocates in the 5′ to 3′ direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage activity of Dna2 was markedly stimulated by the presence of an RNA segment at the 5′-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki fragment. In addition, we demonstrated that the removal of pre-existing initiator 5′-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase δ-catalyzed elongation of the upstream Okazaki fragments. These properties indicate that Dna2 is well suited to remove the Primer RNA on the Okazaki fragment. Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki fragment maturation in conjunction with Fen-1.
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Characterization of the enzymatic properties of the yeast dna2 Helicase/endonuclease suggests a new model for Okazaki fragment processing.
Journal of Biological Chemistry, 2000Co-Authors: Sung-ho Bae, Yeon Soo SeoAbstract:Abstract The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki fragment maturation during lagging strand synthesis. In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes. We found that the Dna2 helicase activity translocates in the 5′ to 3′ direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage activity of Dna2 was markedly stimulated by the presence of an RNA segment at the 5′-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki fragment. In addition, we demonstrated that the removal of pre-existing initiator 5′-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase δ-catalyzed elongation of the upstream Okazaki fragments. These properties indicate that Dna2 is well suited to remove the Primer RNA on the Okazaki fragment. Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki fragment maturation in conjunction with Fen-1.
Hisao Masai - One of the best experts on this subject based on the ideXlab platform.
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eLS - Bacterial Replication Fork: Synthesis of Lagging Strand
eLS, 2010Co-Authors: Taku Tanaka, Hisao MasaiAbstract:How can an antiparallel DNA (deoxyribonucleic acid) strand be duplicated by a DNA polymerase that synthesises DNA in only one direction? This paradox of DNA synthesis on the lagging strand was dissolved by discovery of Okazaki fragments. The major components of the bacterial replication fork include replicative helicase, primase and DNA polymerase. The loading of replicative helicase, DnaB, is the most critical step for assembly of a primosome, a protein complex responsible for duplex unwinding and Primer RNA (ribonucleic acid) synthesis at the replication fork. DNA polymerase may be an asymmetric dimer, each of which may concurrently synthesise leading or lagging strand. Several different modes of primosome assembly have been identified in bacteria. At oriC (origin of chromosome), DnaA-dependent primosome is assembled for initiation of a round of DNA replication, whereas PriA-dependent primosome is assembled at stalled replication forks to facilitate replication restart. Key Concepts: Initiation of DNA replication: DNA replication is initiated by the initiator protein, which specifically recognises and binds to the origin sequence and recruits other primosome components including a DNA helicase. Leading and lagging strands: Leading strand is the one in which the direction of DNA chain elongation and overall fork movement is the same and lagging strand is the one in which they are opposite. Replicative helicase: An enzyme which catalyses continuous unwinding of the parental duplex DNA at the replication fork. Replication fork: The site of DNA replication where two replicating single-stranded DNA separates. Primer RNA: A short stretch of RNA, the 3′-terminus of which is utilised by DNA polymerases for DNA elongation. Primosome: A name given to the protein complex capable of duplex DNA unwinding and Primer RNA synthesis at the replication fork. Stalled replication fork: A replication fork the movement of which is blocked by inteRNAl and exteRNAl ‘replication stress’ including DNA damages and depletion of nucleotide precursors. Replication restart: The process of reassembly of primosome at a stalled replication fork to resume DNA chain elongation. Keywords: replication fork; Okazaki fragment; DNA polymerases; Primer RNA; replication restart
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Frpo: A Novel Single-Stranded DNA Promoter for Transcription and for Primer RNA Synthesis of DNA Replication
Cell, 1997Co-Authors: Hisao Masai, Ken-ichi AraiAbstract:Abstract We describe a novel promoter for E. coli RNA polymerase that functions efficiently only in the form of single-stranded DNA. Derived from the leading region of F plasmid, single-stranded F rpo sequence directs RNA polymerase to initiate transcription at a specific site within F rpo , and this specific transcription is highly stimulated by SSB. Prior denaturation activates transcription from otherwise inactive duplex DNA containing F rpo . Since RNAs synthesized on SSB-coated single-stranded F rpo are efficiently elongated into DNA chains by DNA polymerase III holoenzyme, transcription at F rpo serves also for priming DNA replication. A mode of recognition by RNA polymerase of a unique secondary structure within F rpo is proposed, and possible roles of this novel single-stranded promoter in expression and replication during conjugal transfer of F plasmid are discussed.
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Mechanisms of Primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli
Biochimie, 1996Co-Authors: Hisao Masai, Ken-ichi AraiAbstract:Abstract In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates. These ‘Primers’ are synthesized by various enzymatic mechanisms in Escherichia coli. Studies on Primer RNA synthesis on single-stranded DNA templates containing specific ‘priming signals’ revealed the presence of two distinct modes, ie immobile and mobile priming. The former includes Primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a σ70 subunit. Priming is initiated at a specific site in immobile priming. Novel immobile priming signals were identified from various plasmid replicaons, some of which function in initiation of the leading strand synthesis. The latter, on the other hand, involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication. Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes Primer RNAs at multiple sites. Two distinct primosomes. DnaA-dependent primosome supports normal chromosomal identified, which are differentially utilized for E coli chromosomal replication. Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNAseH activity. In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates Primer RNA synthesis and replication fork progression.
Núria Verdaguer - One of the best experts on this subject based on the ideXlab platform.
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Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-Primer RNA.
The Journal of biological chemistry, 2004Co-Authors: Cristina Ferrer-orta, Armando Arias, Rosa Pérez-luque, Cristina Escarmís, Esteban Domingo, Núria VerdaguerAbstract:Genome replication in picoRNAviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-Primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-Primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.
Sung-ho Bae - One of the best experts on this subject based on the ideXlab platform.
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characterization of the enzymatic properties of the yeast dna2 helicase endonuclease suggests a new model for okazaki fragment processing
Journal of Biological Chemistry, 2000Co-Authors: Sung-ho Bae, Yeon Soo SeoAbstract:Abstract The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki fragment maturation during lagging strand synthesis. In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes. We found that the Dna2 helicase activity translocates in the 5′ to 3′ direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage activity of Dna2 was markedly stimulated by the presence of an RNA segment at the 5′-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki fragment. In addition, we demonstrated that the removal of pre-existing initiator 5′-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase δ-catalyzed elongation of the upstream Okazaki fragments. These properties indicate that Dna2 is well suited to remove the Primer RNA on the Okazaki fragment. Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki fragment maturation in conjunction with Fen-1.
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Characterization of the enzymatic properties of the yeast dna2 Helicase/endonuclease suggests a new model for Okazaki fragment processing.
Journal of Biological Chemistry, 2000Co-Authors: Sung-ho Bae, Yeon Soo SeoAbstract:Abstract The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki fragment maturation during lagging strand synthesis. In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes. We found that the Dna2 helicase activity translocates in the 5′ to 3′ direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage activity of Dna2 was markedly stimulated by the presence of an RNA segment at the 5′-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki fragment. In addition, we demonstrated that the removal of pre-existing initiator 5′-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase δ-catalyzed elongation of the upstream Okazaki fragments. These properties indicate that Dna2 is well suited to remove the Primer RNA on the Okazaki fragment. Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki fragment maturation in conjunction with Fen-1.
Ken-ichi Arai - One of the best experts on this subject based on the ideXlab platform.
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Frpo: A Novel Single-Stranded DNA Promoter for Transcription and for Primer RNA Synthesis of DNA Replication
Cell, 1997Co-Authors: Hisao Masai, Ken-ichi AraiAbstract:Abstract We describe a novel promoter for E. coli RNA polymerase that functions efficiently only in the form of single-stranded DNA. Derived from the leading region of F plasmid, single-stranded F rpo sequence directs RNA polymerase to initiate transcription at a specific site within F rpo , and this specific transcription is highly stimulated by SSB. Prior denaturation activates transcription from otherwise inactive duplex DNA containing F rpo . Since RNAs synthesized on SSB-coated single-stranded F rpo are efficiently elongated into DNA chains by DNA polymerase III holoenzyme, transcription at F rpo serves also for priming DNA replication. A mode of recognition by RNA polymerase of a unique secondary structure within F rpo is proposed, and possible roles of this novel single-stranded promoter in expression and replication during conjugal transfer of F plasmid are discussed.
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Mechanisms of Primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli
Biochimie, 1996Co-Authors: Hisao Masai, Ken-ichi AraiAbstract:Abstract In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates. These ‘Primers’ are synthesized by various enzymatic mechanisms in Escherichia coli. Studies on Primer RNA synthesis on single-stranded DNA templates containing specific ‘priming signals’ revealed the presence of two distinct modes, ie immobile and mobile priming. The former includes Primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a σ70 subunit. Priming is initiated at a specific site in immobile priming. Novel immobile priming signals were identified from various plasmid replicaons, some of which function in initiation of the leading strand synthesis. The latter, on the other hand, involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication. Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes Primer RNAs at multiple sites. Two distinct primosomes. DnaA-dependent primosome supports normal chromosomal identified, which are differentially utilized for E coli chromosomal replication. Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNAseH activity. In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates Primer RNA synthesis and replication fork progression.