Accumulation of ColE1 early replicative intermediates catalyzed by extracts of Escherichia coli dnaG mutant strains

Abstract

To investigate the events occurring at the replication forks during DNA synthesis, we studied the replication of plasmid ColE1 DNA in vivo and in vitro, using strains of Escherichia coli carrying either the dnaG3(Ts) or dnaG308(Ts) mutation. Extracts of both mutant strains supported in vitro DNA synthesis, but the amount of [3H]TMP incorporated into DNA was always less for mutant extracts than for extracts of revertant strains, which were able to grow at 42 degrees C. Sucrose gradient analysis, Southern blot analysis, and electron microscopy showed that mutant extracts synthesize a large number of early replicative intermediates containing one or two (one on each template strand) fragments at the origin of replication and some completed molecules, either open circles or covalently closed circles. The revertant extracts synthesized more completed molecules although the fraction of templates used was about the same, 0.27 for mutant extracts and 0.21 for revertant extracts. Our results show that a mutation in dnaG causes a block in the synthesis of both leading and lagging strands after initiation, which results in the accumulation of early replicative intermediates. The average size of the newly replicated region in the early replicative intermediates is 730 bases as measured from electron micrographs of early replicative intermediates. We conclude that the DnaG protein functions in lagging strand synthesis and may be necessary for the continuation of leading strand synthesis as well.