Replication of the Escherichia coli Chromosome with a Soluble Enzyme System

Semi-conservative DNA synthesis is observed when the isolated, folded E. coli chromosome is supplemented with a DNA-free, soluble enzyme fraction, the four deoxynucleoside 5‧-triphosphates, ATP, and Mg⁺⁺. The DNA synthesized in vitro remains associated with the folded chromosome during sedimentation through neutral sucrose, but is released as small DNA fragments in alkali. Sealing of these replicative intermediates to the chromosome requires the presence of both E. coli DNA polymerase I (EC 2.7.7.7) and DNA ligase (EC 6.5.1.2).