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The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized
Alejandro Serna-Rico1, Margarita Salas, Wilfried J J Meijer
1Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Abstract:
The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.
Insights
The integral membrane protein p16.7 is crucial for bacteriophage phi 29 DNA replication. A soluble variant, p16.7A, binds to single-stranded DNA (ssDNA) in replication intermediates, suggesting a role in attaching these to the host cell membrane.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage phi 29 utilizes a protein-primed replication mechanism.
- Replication intermediates in bacteriophage phi 29 DNA synthesis contain significant single-stranded DNA (ssDNA) regions.
Purpose of the Study:
- To investigate the functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication.
- To characterize the ssDNA-binding properties of a soluble variant of p16.7 (p16.7A).
Main Methods:
- Utilized a soluble variant (p16.7A) lacking the membrane-spanning domain.
- Performed direct and functional analyses to assess ssDNA binding.
- Compared the properties of p16.7A with the known ssDNA-binding protein p5 of phi 29.
Main Results:
- Protein p16.7A exhibits ssDNA-binding activity.
- p16.7A binds specifically to ssDNA within phi 29 DNA replication intermediates.
- p16.7A and p5 possess distinct, non-overlapping functions in phage replication.
Conclusions:
- Protein p16.7 likely functions in anchoring phi 29 DNA replication intermediates to the infected cell membrane.
- Homologues of gene 16.7 in related phages suggest a conserved role for p16.7 in this phage family.