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Bacteriophage phi29 early protein p17 is conditionally required for the first rounds of viral DNA replication
P Crucitti1, J M Lázaro, V Benes
1Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049, Madrid, Spain.
Abstract:
The gene 17 of the Bacillus subtilis phage phi29 is known to be involved in the viral DNA replication in vivo. In this paper, we show that the presence of protein p17 is required when phage infection occurs at a low multiplicity of infection (moi), which is probably the natural condition for infection, but is dispensable at a high moi. Gene 17 has been cloned in an Escherichia coli expression vector and protein p17 purified. A stimulatory effect of protein p17 was demonstrated under in vitro conditions required to amplify phi29 DNA, starting with a low amount of input DNA. We propose that p17, which is synthesized early after infection, is required at the very beginning of the phage amplification, conditions in which a low number of viral DNA molecules enter the host cell, possibly to recruit the limiting amount of initiation factors at the replication origins. Once the infection process is established and the other replication proteins reach optimal concentration, p17 becomes dispensable.
Insights
Bacillus subtilis phage phi29 protein p17 is crucial for viral DNA replication initiation at low multiplicities of infection but not at high multiplicities. This protein aids early phage amplification by recruiting essential factors for DNA replication.
Area of Science:
- Molecular Biology
- Virology
- Microbiology
Background:
- Bacillus subtilis phage phi29 gene 17 encodes protein p17.
- Protein p17 is implicated in viral DNA replication in vivo.
- The natural infection condition is likely a low multiplicity of infection (MOI).
Purpose of the Study:
- To investigate the role of Bacillus subtilis phage phi29 protein p17 in viral DNA replication.
- To determine the dependency of protein p17 during infection at different multiplicities of infection (MOI).
- To elucidate the function of protein p17 in the early stages of phage amplification.
Main Methods:
- Cloning of Bacillus subtilis phage phi29 gene 17 into an Escherichia coli expression vector.
- Purification of protein p17.
- In vitro DNA amplification assays using purified protein p17 under low input DNA conditions.
Main Results:
- Protein p17 is required for phi29 DNA replication at low MOI but dispensable at high MOI.
- Purified protein p17 stimulates phi29 DNA amplification in vitro, especially with limited input DNA.
- Protein p17 is synthesized early after infection and is essential for initiating phage amplification.
Conclusions:
- Protein p17 plays a critical role in the initial stages of phi29 DNA replication under natural infection conditions (low MOI).
- It likely facilitates the recruitment of limiting initiation factors to replication origins.
- Once replication proteins reach optimal concentrations, p17's function becomes redundant.