Protein-primed initiation of phage phi 29 DNA replication
Abstract
We recently reported the development of an in vitro replication system for bacteriophage phi 29 DNA. We have used this system for the isolation of replication activity associated with gene 3 protein (terminal protein) from phi 29-infected Bacillus subtilis cells. We utilized two assay systems: (i) DNA replication dependent on phi 29 DNA with the 5' end covalently linked to terminal protein (DNA-protein) and (ii) the formation of complex between the terminal protein and dAMP. The DNA-replication and the complex-forming activities were purified together through all steps. The complex of terminal protein and dAMP formed in the purified fraction was shown to serve as an effective primer for successive chain elongation in the presence of dNTPs by a pulse-chase experiment. The protein fraction purified from cells infected with a temperature-sensitive phi 29 mutant in gene 3 was thermolabile compared to the wild-type activity in the assay system for complex formation. This shows that the purified fraction having replication activity includes the gene 3 product of phi 29. Both the DNA replication and the complex formation activities are highly specific for phi 29 DNA-protein as template. The product analysis of elongated DNA revealed t...Continue Reading
References
Citations
Effect of ultraviolet radiation on the Bacillus subtilis phages SPO2, SPP1 and phi 29 and their DNAs
Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase
Related Concepts
Related Feeds
Bacteriophage: Phage Therapy
Phage therapy uses bacterial viruses (bacteriophages) to treat bacterial infections and is widely being recognized as an alternative to antibiotics. Here is the latest research.