Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly.
Identifieur interne : 001625 ( Main/Exploration ); précédent : 001624; suivant : 001626Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly.
Auteurs : Aleksandra Wawrzycka [Pologne] ; Marta Gross [Pologne] ; Anna Wasaznik [Pologne] ; Igor Konieczny [Pologne]Source :
- Proceedings of the National Academy of Sciences of the United States of America [ 1091-6490 ] ; 2015.
Descripteurs français
- KwdFr :
- Adenosine triphosphatases (métabolisme), Complexes multienzymatiques (métabolisme), DNA-directed DNA polymerase (métabolisme), Dichroïsme circulaire, DnaB Helicases (métabolisme), Escherichia coli (enzymologie), Liaison aux protéines, Matrices (génétique), Nucléoprotéines (métabolisme), Origine de réplication, Plasmides (métabolisme), Protéines Escherichia coli (métabolisme), Protéines mutantes (métabolisme), Réplication de l'ADN, Sous-unités de protéines (métabolisme).
- MESH :
- enzymologie : Escherichia coli.
- métabolisme : Adenosine triphosphatases, Complexes multienzymatiques, DNA-directed DNA polymerase, DnaB Helicases, Nucléoprotéines, Plasmides, Protéines Escherichia coli, Protéines mutantes, Sous-unités de protéines.
- Dichroïsme circulaire, Liaison aux protéines, Matrices (génétique), Origine de réplication, Réplication de l'ADN.
English descriptors
- KwdEn :
- Adenosine Triphosphatases (metabolism), Circular Dichroism, DNA Replication, DNA-Directed DNA Polymerase (metabolism), DnaB Helicases (metabolism), Escherichia coli (enzymology), Escherichia coli Proteins (metabolism), Multienzyme Complexes (metabolism), Mutant Proteins (metabolism), Nucleoproteins (metabolism), Plasmids (metabolism), Protein Binding, Protein Subunits (metabolism), Replication Origin, Templates, Genetic.
- MESH :
- chemical , metabolism : Adenosine Triphosphatases, DNA-Directed DNA Polymerase, DnaB Helicases, Escherichia coli Proteins, Multienzyme Complexes, Mutant Proteins, Nucleoproteins, Protein Subunits.
- enzymology : Escherichia coli.
- metabolism : Plasmids.
- Circular Dichroism, DNA Replication, Protein Binding, Replication Origin, Templates, Genetic.
Abstract
Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β- and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined.
DOI: 10.1073/pnas.1504926112
PubMed: 26195759
Affiliations:
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wicri:level="1"><nlm:affiliation>Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland.</nlm:affiliation><country xml:lang="fr">Pologne</country><wicri:regionArea>Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk</wicri:regionArea><wicri:noRegion>80-822 Gdansk</wicri:noRegion></affiliation></author><author><name sortKey="Gross, Marta" sort="Gross, Marta" uniqKey="Gross M" first="Marta" last="Gross">Marta Gross</name><affiliation wicri:level="1"><nlm:affiliation>Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk, Poland.</nlm:affiliation><country xml:lang="fr">Pologne</country><wicri:regionArea>Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, 80-822 Gdansk</wicri:regionArea><wicri:noRegion>80-822 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Triphosphatases</term><term>DNA-Directed DNA Polymerase</term><term>DnaB Helicases</term><term>Escherichia coli Proteins</term><term>Multienzyme Complexes</term><term>Mutant Proteins</term><term>Nucleoproteins</term><term>Protein Subunits</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Plasmids</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Adenosine triphosphatases</term><term>Complexes multienzymatiques</term><term>DNA-directed DNA polymerase</term><term>DnaB Helicases</term><term>Nucléoprotéines</term><term>Plasmides</term><term>Protéines Escherichia coli</term><term>Protéines mutantes</term><term>Sous-unités de protéines</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Circular Dichroism</term><term>DNA Replication</term><term>Protein Binding</term><term>Replication Origin</term><term>Templates, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Dichroïsme circulaire</term><term>Liaison aux protéines</term><term>Matrices (génétique)</term><term>Origine de réplication</term><term>Réplication de l'ADN</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β- and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined. </div></front></TEI><affiliations><list><country><li>Pologne</li></country></list><tree><country name="Pologne"><noRegion><name sortKey="Wawrzycka, Aleksandra" sort="Wawrzycka, Aleksandra" uniqKey="Wawrzycka A" first="Aleksandra" last="Wawrzycka">Aleksandra Wawrzycka</name></noRegion><name sortKey="Gross, Marta" sort="Gross, Marta" uniqKey="Gross M" first="Marta" last="Gross">Marta Gross</name><name sortKey="Konieczny, Igor" sort="Konieczny, Igor" uniqKey="Konieczny I" first="Igor" last="Konieczny">Igor Konieczny</name><name sortKey="Wasaznik, Anna" sort="Wasaznik, Anna" uniqKey="Wasaznik A" first="Anna" last="Wasaznik">Anna Wasaznik</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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