E. coli minichromosome replication in vitro and in vivo: comparative analyses of replication intermediates

Abstract

The process of replication of Escherichia coli minichromosomes was examined by following the intermediates formed in vitro and in vivo. Replication initiated on a supercoiled closed circular (CC) monomer, proceeded rapidly to a late but incomplete stage in polymerization (the LC form) in both systems, passed more slowly through a series of open and closed circular catenated dimers with varying extents of intertwining between the monomer units, and then yielded, after decatenation, the supercoiled CC monomer. The replication patterns of two different minichromosomes were similar, although the LC form and the multiply intertwined dimers were much more evident in the smaller pAL4 than in pAL2. The same basic replication scheme was seen in vitro and in vivo but completion of polymerization and processing of the dimers were slower in vitro. Some radioactivity was detected in OC monomer early during replication, consistent with occasional decatenation of LC structures to produce OC molecules which then completed replication to form CC molecules. However, progression to CC catenated dimers prior to formation of CC monomers represented the major replication pathway.