Bacteriophage T7 gene 4 protein and DNA polymerase of the phage were used to study the viral strand synthesis of bacteriophage fd in vitro. Cleavage of supercoiled phage fd replicative form (RF) by fd gene 2 protein produced a nick at a specific site in the viral strand. The cleaved double-stranded DNA was unwound by T7 gene 4 protein and T7 DNA polymerase and the 3' end of the nicked strand simultaneously extended according to the rolling circle mechanism. After a complete round of DNA synthesis fd gene 2 protein cleaved the viral strand presumably at the same site, where the endonuclease cuts fd R F I, and subsequently sealed the single-stranded linear DNA into a circle. The reaction products were analyzed by velocity sedimentation, gel electrophoresis and electron microscopy. Most of the single-stranded DNA synthesized was circular. No host proteins were required for the formation of the single-stranded circles. Strand switching of the T7 DNA polymerase indicated by doublestranded tails of the rolling circle structures reduced the yield of viral single-stranded circles in this enzyme system.

The gene for Escherichia coli rep helicase (rep protein) was subcloned in a pBR plasmid and the protein overproduced in cells transformed with the hybrid DNA. The effect of purified enzyme on strand unwinding and DNA replication was investigated by electron microscopy. The templates used were partial duplexes of viral DNA from bacteriophage fd: :Tn5 and reannealed DNA from bacteriophage Mu. The experiments with the two DNA species show DNA unwinding uncoupled from replication. The single-stranded phage fd: :Tn5 DNA with the inverted repeat of transposon Tn5 could be completely replicated in the presence of the E. coli enzymes rep helicase, DNA binding protein I, RNA polymerase and DNA polymerase I11 holoenzyme. A block in the unwinding step increases secondary initiation events in single-stranded parts of the template, as DNA polymerase I11 holoenzyme cannot switch across the stem structure of the transposon.The Escherichia coli gene rep has been implicated with replication of small bacteriophages [l]. The enzymatic involvement of rep helicase in strand unwinding has been shown in vitro for replication of double-stranded phage 4x174 [2] and phage fd DNA [3].The action of rep helicase in the fork depends on ATP or dATP as energy source [4]. The polarity of unwinding by rep helicase is opposite to the direction of strand separation found for other E. coli DNA helicases [5]. The rep helicase separates the strands in the direction of DNA replication on the leading strand. A general method to assay the action of DNA helicases is the degradation of single-stranded DNA generated by SI-endonuclease [6]. Partially unwound duplex DNA cannot be assayed by this method, as the strands will renature before completion of endonucleolytic degradation or endonucleolytic digest of partial duplexes cannot be distinguished from degradation of completely separated strands. However, electron microscopy is a convenient tool to study partial denaturation of double-stranded DNA [7].The E. coli DNA helicases I, I1 and 111 require a DNA duplex to be partially single-stranded in order to unwind DNA [8]. E. coli rep helicase can initiate DNA unwinding at a nick if a helper protein like gene 2 protein for phage fd [3] or gene A protein for phage 4x174 DNA is present [9]. In the absence of these phage-specific proteins, unwinding by rep helicase also depends on single-stranded regions in the duplex DNA [lo]. The enzyme might also be involved in replication of the host chromosome. Defects in the coding gene results in increased fork initiation [ll]. The rep mutation does not seem compatible with the non-functional uvrD gene product (DNA helicase 11) [12]. It has therefore been assumed that E. coli rep helicase and DNA helicase I1 simultaneously unwind the two strands in a replication fork.Here we will describe electron microscopical investigations on enzymatic strand separation by E. coli rep helicase in the presence of E. coli DNA binding protein I. It will be shown that DNA replication by DNA polymerase I11 holoenzyme on a Abbreviation. RF, r...

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