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Replication terminus for DNA polymerase I during initiation of pAM beta 1 replication: role of the plasmid-encoded
L Jannière1, V Bidnenko, S McGovern
1Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy-en-Josas, France. janniere@biotec.jouy.inra.fr
Abstract:
Replication of plasmid pAM beta 1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D-loop structures containing the nascent leading strand paired to its template, and that D-loop extension is arrested approximately 230 bp from the initiation site of DNA synthesis in the presence of the plasmid-encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D-loop replication intermediates carry a single-stranded primosome-assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands.
Insights
Plasmid DNA replication initiation by DNA polymerase I (Pol I) is halted by a resolvase enzyme, creating D-loop structures. This interaction promotes replisome machinery takeover for efficient DNA synthesis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Plasmid pAM beta 1 replication involves DNA polymerase I (Pol I) for initiation and DNA polymerase III holoenzyme for completion.
- Understanding replication initiation mechanisms is crucial for genetic stability and biotechnological applications.
Purpose of the Study:
- To investigate the mechanism of replication arrest during plasmid pAM beta 1 DNA synthesis.
- To elucidate the role of the plasmid-encoded resolvase in regulating replication fork progression.
Main Methods:
- In vitro and in vivo assays were employed to study DNA replication intermediates.
- Analysis of D-loop structures and their interaction with DNA polymerases and resolvase.
Main Results:
- Replication initiation generates D-loop structures, with extension arrested ~230 bp from the initiation site.
- The arrest is attributed to a collision between Pol I and the bound resolvase.
- Arrested D-loops contain primosome-assembly sites.
Conclusions:
- The replication arrest mechanism limits Pol I activity and facilitates replisome engagement.
- This process ensures efficient and coordinated leading and lagging strand synthesis during plasmid replication.