DNA replication of bacteriophage θ29: Characterization of the intermediates and location of the termini of replication

Abstract

Replicative intermediates of the linear duplex of bacteriophage θ29 have been isolated and partially characterized. Replicative DNA, pulse-labeled with [ 3H]thymidine, behaves in neutral sucrose gradient sedimentation and agarose gel electrophoresis as mature θ29 DNA but with a portion of the replicative DNA being of slightly larger size. The faster-sedimenting DNA exhibits an increased buoyant density. In alkaline sucrose gradients, the denatured strands sediment as unit length monomers or smaller fragments. No strands longer than genome length were observed. Sensitivity of the pulse-labeled DNA to S1 nuclease and behavior on benzoylated-naphthoylated DEAE-cellulose (BND-cellulose) chromatography indicate that the intermediates are characterized by extensive single-stranded regions. Intracellular DNA was fractionated on BND-cellulose into completed (double stranded) molecules and replicating (partially single stranded) molecules. Restriction enzyme analysis of the distribution of radioactivity in pulse-labeled completed molecules indicates that termini of replication (most highly labeled regions) are at both ends of the DNA. Electron microscopic analysis of replicating molecules reveals two primary types of intermediates: (1) linear branched molecules with one single-stranded arm and (2) unbranched linear molecules with a single-stranded region extending from one end. These results suggest that θ29 DNA replicates by an asymmetric displacement mechanism. Thus, synthesis is initiated at one end and proceeds in the 5′ to 3′ direction with concomitant displacement of the parental strand of the same polarity. Synthesis of the complementary strand is initiated at the 3′ end of the displaced parental strand. A role for the covalently linked terminal protein in initiation of DNA synthesis is suggested.