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Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase
1Centro de Biologia Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Universidad Autonoma, Canto Blanco, Madrid, Spain.
Abstract:
Phage phi29 from Bacillus subtilis is a paradigm of the protein-primed replication mechanism, in which a single-subunit DNA polymerase is involved in both the specific protein-primed initiation step and normal DNA elongation. To start phi29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each phi29 DNA end. The results shown in this paper demonstrate that the DNA polymerase-primer TP heterodimer is not dissociated immediately after initiation. On the contrary, there is a transition stage in which the DNA polymerase synthesizes a five nucleotide-long DNA molecule while complexed with the primer TP, undergoes some structural change during replication of nucleotides 6-9, and finally dissociates from the primer protein when nucleotide 10 is inserted onto the nascent DNA chain. This behaviour probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation. The significance of such a limiting transition stage is supported by the finding of abortive replication products consisting of the primer TP linked up to eight nucleotides, detected during in vitro replication of phi29 TP-DNA particularly under conditions that decrease the strand-displacement capacity of phi29 DNA polymerase.
Insights
Phage phi29 DNA polymerase forms a complex with the terminal protein (TP) primer during initiation. This complex undergoes a transition stage before dissociating, ensuring efficient DNA elongation.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Phage phi29 utilizes a unique protein-primed DNA replication mechanism.
- A single-subunit DNA polymerase is central to both initiation and elongation.
- Initiation involves the DNA polymerase priming synthesis with the viral terminal protein (TP).
Purpose of the Study:
- To investigate the dissociation dynamics of the DNA polymerase-TP complex during phi29 DNA replication initiation.
- To understand the role of the polymerase-primer interaction in the transition stage of replication.
- To elucidate the functional significance of the polymerase's requirement for a minimum DNA primer length.
Main Methods:
- In vitro replication assays using phi29 TP-DNA.
- Analysis of replication intermediates and abortive products.
- Characterization of the DNA polymerase-TP complex during early synthesis stages.
Main Results:
- The DNA polymerase-TP heterodimer does not dissociate immediately after initiation.
- A transition stage exists where the polymerase synthesizes a 5-nucleotide DNA while complexed with TP.
- Dissociation occurs upon insertion of the 10th nucleotide, suggesting a minimum primer length requirement.
Conclusions:
- The DNA polymerase-TP complex undergoes a critical transition phase before dissociation.
- This transition ensures the polymerase has a sufficiently long DNA primer for efficient elongation.
- Abortive replication products support the existence and significance of this transition stage.