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Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication
Abstract:
The phage phi 29 protein p2, required for the formation of the protein p3-dAMP initiation complex, has been purified from Escherichia coli cells harboring a gene 2-containing recombinant plasmid. The purified protein p2, of molecular weight 68,000, had a specific DNA polymerase activity that elongated the p3-dAMP initiation complex when phi 29 DNA-protein p3 was used as template. In addition, the purified protein p2 was active in catalyzing the initiation reaction when complemented with phi 29 mutant sus2-infected Bacillus subtilis or plasmid-containing E. coli extracts providing protein p3, in the presence of phi 29 DNA-protein p3 as template. However, when purified protein p3 was used in the complementation assay, a very low amount of initiation complex was formed; addition of extracts from uninfected B. subtilis or E. coli strongly stimulated the initiation reaction, indicating that, in addition to proteins p2 and p3 and the phi 29 DNA-protein p3 template, some host factor(s) is required for the formation of the p3-dAMP initiation complex. The results show that phage phi 29 encodes a DNA polymerase that is required at the initiation step of protein-primed DNA synthesis.
Insights
Phage phi 29 protein p2 is a DNA polymerase essential for initiating protein-primed DNA synthesis. This protein, along with protein p3 and host factors, is crucial for forming the initiation complex during DNA replication.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Phage phi 29 utilizes a protein-primed mechanism for DNA replication.
- Protein p2 is known to be involved in the initiation complex formation.
Purpose of the Study:
- To purify and characterize phage phi 29 protein p2.
- To investigate the role of protein p2 in the initiation of DNA synthesis.
Main Methods:
- Purification of phage phi 29 protein p2 from recombinant Escherichia coli.
- Assaying DNA polymerase activity of purified protein p2.
- Complementation assays using bacterial extracts and purified proteins.
Main Results:
- Purified protein p2 exhibits DNA polymerase activity, elongating the protein p3-dAMP initiation complex.
- Protein p2 catalyzes initiation reactions in the presence of protein p3 and phi 29 DNA-protein p3 template.
- Host factors from Bacillus subtilis and Escherichia coli are required for efficient initiation complex formation.
Conclusions:
- Phage phi 29 encodes a DNA polymerase (protein p2) essential for the initiation step of protein-primed DNA synthesis.
- The formation of the initiation complex requires proteins p2, p3, phi 29 DNA-protein p3 template, and host factors.