Semiconservative replication

Abstract: The colicinogenic factor E(1) (Col E(1)) in Escherichia coli continues to replicate by a semiconservative mechanism in the presence of chloramphenicol (CAP) for 10 to 15 hr, long after chromosomal deoxyribonucleic acid (DNA) synthesis has terminated. Following CAP addition, the rate of synthesis of plasmid DNA gradually increases to an extent dependent on the medium employed. Within 2 to 4 hr after the addition of CAP, replication in a glucose-Casamino Acids medium approaches a maximum rate representing approximately eight times an average rate which would be required for a net doubling of DNA per cell in one generation. The number of copies of Col E(1) DNA molecules that accumulate under these conditions approaches about 3,000 copies per cell, representing a 125-fold increase over the normal level of 24 copies per cell. The system is particularly convenient for studying the mechanism of DNA replication.

Abstract: SUMMARY An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.