Abstract
Using purified enzymes, double strand replication of phage fd DNA has been dissected into several intermediate steps. (i) Phage fd gene 2 protein cleaves supercoiled phage fd replicative form at a specific site in the viral strand (Meyer, T. F., Geider, K., Kurz, C., and Schaller, H. (1979) Nature 278, 365-367). (ii) Relaxed covalently closed circular replicative form DNA which is also formed by gene 2 protein as a side product in the initiation reaction preceding replication is converted into supercoils by DNA gyrase. (iii) The enzyme forms a noncovalent complex at the generated nick that is necessary for initiation of subsequent unwinding. (iv) The Escherichia coli rep helicase (rep protein) and E. coli DNA binding protein I unwind the double-stranded DNA. (v) Concomitant DNA replication by E. coli DNA polymerase III holoenzyme results in the formation of rolling circle intermediates. The double-stranded core of the rolling circle remains in an open form, thus allowing continued synthesis during several rounds of replication. (vi) Processing of replicated viral DNA can be subdivided into the cleavage and the circularization of viral single strands. Comparative studies of fd and phi X174 replication in vitro have revealed differences in the kinetics of individual steps besides an apparent contrast in the conformation of rolling circle intermediates in the electron microscopy where fd DNA features extended tails rather than looped-back structures observed for phi X174 DNA.
Highlights
Double strand replicationf gene A protein, it starts the unwinding of the phage double phage fd DNA has been dissected into several inter- strands at a specificnick (14, 15), but it requires a singlemediate steps. (i) Phage fd gene 2 protein cleaves su- stranded region whenit separates strandswithout t& protein percoiled phage fd replicative form at a specisficte in (16).The function of rep helicase is similar to the viral strand
H. (1979) Nature 278,365-367). (ii)Relaxed a sequence of single-stranded DNA in front of the doublecovalently closed circular replicative forDmNA which is formed by gene 2 protein as a side product in theinitiationreactionprecedingreplication is converted into supercoils byDNA gyrase. (iii)“he enzyme forms a noncovalent complex at the generated nick that is necessary for initiation of subsequent unwinding. (iv) TheEscherichia coli rep helicase and E. coli DNA binding proteinI unwind the doublestranded DNA.(v) Concomitant DNA replication byE
In contrast to phage $X174 gene A protein (2) the analogous phage fd gene2 protein is not tightly attached to the5’-end of the cleaved strand (21).It was, expected that rolling circle structures of fd DNA may differ fromthose observed as coli DNA polymerase III holoenzymeresults in the phage 4x174 replication intermediates
Summary
From the Max-Planck-Institut fumr edizinische Forschung, Abteilung MolekulareBiologie, Jahnstrasse 29,06900 Heidelberg, Federal Republic of Germany. (ii)Relaxed a sequence of single-stranded DNA in front of the doublecovalently closed circular replicative forDmNA which is formed by gene 2 protein as a side product in theinitiationreactionprecedingreplication is converted into supercoils byDNA gyrase. In contrast to phage $X174 gene A protein (2) the analogous phage fd gene protein is not tightly attached to the5’-end of the cleaved strand (21).It was, expected that rolling circle structures of fd DNA may differ fromthose observed as coli DNA polymerase III holoenzymeresults in the phage 4x174 replication intermediates. The double- detailed analysis of the involvement of the individual comstranded core of the rolling circle remains in an open ponents of the replication system on intermediate stages of form, allowing continued synthesis during severaunl winding and replication of phage fd duplex DNA
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