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DNA Synthesis Initiated At OriC: In Vitro Replication Reactions.
Published 1995 · Biology, Medicine
Publisher Summary In vitro replication initiated at the Escherichia coli chromosomal origin (oriC) can occur in a precisely prepared cell extract or it can be reconstituted with purified proteins. The development of these systems was possible following the identification of oriC and the construction of oriC-containing plasmids. In vivo , these plasmids act as minichromosomes in that their replication is initiated synchronously with that of the cell chromosome and with similar requirements. Behaving as such, these plasmids can serve as templates in vitro to study replication at oriC. Another major advance in the development of systems to replicate oriC in vitro is the preparation of an extract containing all of the required factors and lacking inhibitors. Combined with knowledge gained from studying replication in other systems such as bacteriophage φX174, replication in the crude extract permitted the identification, isolation, and characterization of the numerous components necessary to reconstitute oriC DNA replication. Replication in cell extracts and in systems of defined components has many physiological features. It requires the addition of template DNA, which contains the oriC sequence, and replication is initiated at or near oriC. It is dependent on certain replication proteins that are identified as important for chromosomal replication in vivo . Included in these is DnaA, a protein that is implicated genetically as playing a central role in initiating replication at oriC.