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Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the
1Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Abstract:
The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase. We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.
Insights
Bacteriophage phi29 protein p1 interacts with the terminal protein (TP) to regulate DNA replication. Truncated p1 proteins can inhibit initiation complex formation, suggesting a role in controlling viral DNA synthesis.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage phi29 replication protein p1 (p1) is membrane-associated in infected cells.
- The C-terminal 52 amino acids of p1 are sufficient for protofilament sheet assembly.
Purpose of the Study:
- To investigate the interaction of truncated p1 proteins with the primer terminal protein (TP).
- To elucidate the role of p1 in the initiation of phi29 DNA replication.
Main Methods:
- Chemical cross-linking experiments to study protein interactions.
- In vitro and in vivo assays to assess DNA replication rates and complex formation.
- Analysis of truncated p1 protein variants (p1DeltaC43 and N-terminal truncations).
Main Results:
- p1DeltaC43, a C-terminally truncated p1, interacts with TP in vitro.
- Both p1 and p1DeltaC43 inhibit phi29 DNA replication in vivo in a dose-dependent manner.
- N-terminally truncated p1 proteins interfere with TP.dAMP initiation complex formation, which is suppressed by increased TP or phi29 DNA polymerase concentrations.
Conclusions:
- A model is proposed where the viral replisome attaches to a membrane-associated p1 structure for initiation of in vivo phi29 DNA replication.
- Protein p1 plays a crucial role in regulating the initiation of phi29 DNA replication through interactions with TP and potentially membrane structures.