Analysis of replicative intermediates produced during bacteriophage φ29 DNA replication in vitro

Abstract

Replication of bacteriophage φ29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro φ29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded φ29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length φ29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a singlestranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro φ29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded φ29 DNA molecules were synthesized. In addition, replication of recombinant φ29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded φ29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.