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).Study of the replication of Escherichia coli DNA has been complicated both by the need for coordinated interplay of many enzymes and by the fragile nature of the template. For these reasons E. coli DNA replication has not been successfully reconstructed in solution. DNA synthesis observed in cell-free extracts (1-7) represents catalytic actions of the DNA polymerases (1, 5, 7-10), but lacks many of the important features of replication. Moses and Richardson (11) showed that E. coli cells rendered permeable by toluene to low-molecular-weight precursors are able to replicate their DNA; however, the cells remained impermeable to proteins and thus to fractionation and dissection of the enzymatic steps of replication. Schaller et al. (12) demonstrated that replication could proceed in cells lysed on cellophane discs; however, this system appears to require the tight complex of macromolecular components of the bacterial cell, rendering its fractionation extremely difficult.We were encouraged to attempt the replication of DNA in a soluble system by two recent advances. First, a special highspeed supernatant from gently lysed cells (13,14) has been shown to contain the active gene products (14, 15) defined genetically to be essential for E. coli DNA synthesis (the dnaA, B, C, E, and G proteins) (16) as well as other replicationproteins not yet defined by genetic lesions (17). Second, a method has been developed whereby E. coli spheroplasts yield a nonviscous preparation of intact, folded, and supercoiled chromosomes (18,19).These isolated folded chromosomes behave like compact particles, and sediment either as free, folded chromosomes at 1300-2200 S or at 3000-4000 S in a membrane-attached form (20,21). The physical properties of these extracted particles vary with the cell's physiological state: replicating chromosomes isolated at 10°-15°in 1 M NaCl remain associated with the bacterial envelope, whereas nonreplicating chromosomes are released membrane-free (20). The size distribution of the chromosomes also reflects their growth during replication (19).The exploitation of these techniques has allowed us to demonstrate and characterize the unique DNA synthesis that occurs when the folded chromosomes are combined with a DNA-free, soluble enzyme system. As reported below, this DNA synthetic reaction contains many of the features embodied in in vivo chromosome replication. Henry; DNA polymerase I (DNA nucleotidyl transferase, EC 2.7.7.7, 3 mg/ml, 18,000 units/mg) a gift from Dr. D. Brutlag;...

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