Structure of protein-containing replicative intermediates of Bacillus subtilis phage φ29 DNA

Abstract

Protein-containing φ29 DNA replicative intermediates were isolated from extracts of phage-infected Bacillus subtilis. After allowing protein-protein interaction in vitro, analysis with the electron microscope showed the presence of circular type I molecules (unit-length duplex DNA with one single-stranded branch at a random position) and circular type II molecules (unit-length DNA with one double-stranded region and a single-stranded region extending a variable distance from one end). The circular replicative intermediates obtained are consistent with the presence of the terminal protein at the ends of the parental and daughter DNA strands, both in type I and type II molecules. The presence of protein in the replicating molecules was confirmed by retention of the labeled DNA on cellulose nitrate filters. By polyacrylamide gel electrophoresis, the protein bound to the DNA molecules was shown to be the terminal protein, p3. The results obtained support a model in which protein p3 acts as a primer in the initiation of φ29 DNA replication.