Replication of Bacteriophage φ 29 DNA in vitro: The Roles of Terminal Protein and DNA Polymerase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1984-09, Vol.81 (17), p.5374-5378
• Main Authors: Watabe, Kounosuke, Leusch, Mark, Ito, Junetsu
• Format: Article
• Language: English
• Subjects: Adenoviruses; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacteriophages; Bacteriophages - enzymology; Bacteriophages - genetics; Cell extracts; Chromatography; DNA; DNA Replication; DNA-Directed DNA Polymerase - isolation & purification; DNA-Directed DNA Polymerase - metabolism; Enzymes; Escherichia coli - genetics; Gels; Genes, Viral; Monomers; Plasmids; Viral Proteins - isolation & purification; Viral Proteins - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.81.17.5374

φ 29 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP). This initiation reaction system has been reconstituted from two phage-encoded proteins, the terminal protein and DNA polymerase. The φ 29 DNA polymerase was purified from phage-infected cells by using poly(dA)· p(dT)12-18as an assay template. The purified polymerase has an apparent molecular mass of 68 kDa in its native form and it appears to function as a monomer. The terminal protein was purified to homogeneity from Escherichia coli cells harboring a cloned plasmid that contained a φ 29 gene 3 segment. The molecular mass of the purified terminal protein was about 30 kDa in both the denatured and the native form. The protein apparently functions as a monomer. When the terminal protein and DNA polymerase were incubated in the presence of dATP, Mg2+, and φ 29 DNA-protein as template, the terminal protein bound covalently to dAMP. This reaction did not require ATP. In addition, these two purified fractions catalyzed DNA chain elongation from both ends of φ 29 DNA, yielding the expected 9- to 12-base fragment when assayed in the presence of 2′,3′-dideoxycytidine triphosphate. These results indicate that φ 29 DNA polymerase catalyzes formation of the terminal protein-dAMP complex and can also catalyze chain elongation at least 9-12 bases from both ends of φ 29 DNA.