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In prokaryotes like E. coli, DNA replication begins at the origin of replication, called OriC. Initiator proteins bind to OriC and initiate DNA separation. Helicase is then loaded onto each strand to unwind the DNA further and form a replication bubble, which contains two replication forks that proceed bidirectionally around the circular chromosome.

Single-strand DNA binding proteins, or SSBs, stabilize the unwound DNA strands as the replication fork travels along the chromosome. These proteins prevent the separated template strands from re-annealing or forming secondary structures, maintaining them in a form accessible to DNA polymerases for synthesis of leading strand and lagging strand synthesis.

As DNA unwinds at the replication fork, the DNA ahead becomes overwound and supercoiled, creating torsional strain. Type one topoisomerase enzymes relieve this stress by creating a nick in one strand of the double-stranded DNA phosphate-sugar backbone, allowing the other strand to pass through, then ligating the DNA back together to restore integrity.

DNA polymerase can only synthesize DNA in the 5' to 3' direction. On the lagging strand template, which runs 3' to 5' toward the replication fork, synthesis must occur in short segments called Okazaki fragments, typically 1,000–2,000 base pairs long, rather than continuously like on the leading strand.

E. coli DNA polymerase III, responsible for the majority of replication, can polymerize approximately 1,000 base pairs per second. This remarkable speed allows the replication machinery at the two replication forks to duplicate the entire E. coli chromosome of 4.6 million base pairs in roughly 40 minutes under optimal conditions.

Under favorable growth conditions, E. coli can divide every 20 minutes, faster than the 40 minutes needed for complete genome replication. Bacteria solve this by initiating another round of DNA replication from the origin of replication before the first round completes, so daughter cells receive a chromosome already undergoing replication and are prepared to divide again quickly.

As replication proceeds bidirectionally around the circular prokaryotic chromosome, the two replication forks eventually meet at termination or ter sites where DNA replication completes. The resulting daughter DNA molecules each contain one parental strand and one newly synthesized strand, following the semi-conservative model of DNA replication.