In vitro initiation of bacteriophage Φ29 and M2 DNA replication: Genes required for formation of a complex between the terminal protein and 5′dAMP

Abstract

Cell-free extracts prepared from Φ29 and M2-infected Bacillus subtilis cells catalyse the formation of complexes between terminal protein and [α-³²P]-dAMP in the presence of [α-³²P]-dATP, MgCl₂, ATP, and phage DNA with terminal protein covalently linked at both the 5′ends. The complex formation does not take place when proteinase K-treated DNA is added or when uninfected extract is used. The Φ29 complex thus formed is smaller than the M2 complex, primarily due to the different molecular weights of the respective terminal proteins. Extracts prepared from cells infected with suppressor-sensitive mutants of genes 2 or 3 of Φ29 or genes G or E of M2 do not support complex formation. When the pair of extracts of Φ29 or M2-infected cells are mixed, however, formation of the complex takes place as a result of in vitro complementation. These results indicate that the complex formation observed in vitro reflects in vivo initiation of phage DNA replication. The product of gene 2 of Φ29 may be the enzyme that catalyses formation of the complex.