Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase

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Abstract
Phage φ29 from Bacillus subtilis is a paradigm of the protein‐primed replication mechanism, in which a single‐subunit DNA polymerase is involved in both the specific protein‐primed initiation step and normal DNA elongation. To start φ29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each φ29 DNA end. The results shown in this paper demonstrate that the DNA polymerase–primer TP heterodimer is not dissociated immediately after initiation. On the contrary, there is a transition stage in which the DNA polymerase synthesizes a five nucleotide‐long DNA molecule while complexed with the primer TP, undergoes some structural change during replication of nucleotides 6–9, and finally dissociates from the primer protein when nucleotide 10 is inserted onto the nascent DNA chain. This behaviour probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation. The significance of such a limiting transition stage is supported by the finding of abortive replication products consisting of the primer TP linked up to eight nucleotides, detected during in vitro replication of φ29 TP‐DNA particularly under conditions that decrease the strand‐displacement capacity of φ29 DNA polymerase.