Signals in the φ29 DNA-terminal protein template for the initiation of phage φ29 DNA replication

Abstract

The protein-free terminal fragments HindIII B and L, from the left and right ends of φ29 DNA, respectively, but not internal fragments of similar size, were active as templates in the formation of the p3-dAMP initiation complex in an in vitro system containing purified φ29 terminal protein p3 and DNA polymerase p2, although the activity was lower than that obtained with the φ29 DNA-p3 complex. These results indicate the existence of specific sequences at the ends of φ29 DNA that allow the initiation of φ29 DNA replication. The template activity of the protein-free terminal fragments was size dependent. The protein-free single strands of the HindIII L fragment were much less active than the corresponding double-stranded fragment. Terminal protein-DNA complexes of phages PZA and φ15, with a terminal protein closely related to the φ29 protein p3, were more active as templates in the initiation reaction with the purified φ29 proteins than the corresponding protein-free DNAs, as it happens in the case of φ29 However, the terminal protein-DNA complexes of phages Nf, B103, and GA-1, with a terminal protein less related or unrelated to the φ29 protein p3, were essentially inactive and became active after removal of the parental terminal protein. These results strongly suggest that the parental terminal protein is the major signal in the template for the initiation of φ29 DNA replication.