Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events.
Bottom Line: To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin.We also show that the presence of a correctly positioned displaced strand is important because origins with 5' or 3' ssDNA regions have very low activity.We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.
Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.Show MeSH
Mentions: A model that takes into account the results presented in this article, together with previous information, is shown in Figure 8. In slow motion, a possible order of events for Φ29 early replication would be as follows. The parental TP and the p6 complex bound at the end of the Φ29 genome would recruit the TP/DNA polymerase heterodimer to the origin. Then, the unwinding of the origin would start, possibly helped by protein p6, and the TP/DNA polymerase complex would bind to the template strand and recognize the AAA at the 5′ end of the displaced strand, and this complex probably also interacts with sequences from the fourth bp on. At the same time, the Nt domain would stabilize the complex by binding to dsDNA regions of the origin in a non-sequence specific fashion, and the initiating nucleotides would start being incorporated. Further unwinding of the origin would favor the binding of the TP/DNA polymerase complex to the displaced strand and the continuation of replication through the transition phase. At that point a thermodynamic barrier would have to be passed by continuing the unwinding of the DNA strands at the same time as it happens the dissociation of the DNA polymerase from the TP utilized as primer. These events are probably driven by the nascent DNA chain. After the passage from transition to elongation, the DNA polymerase, now dissociated from the TP, would continue in a processive, strand-displacing, mode to replicate the whole phage genome.
Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.