Several principles govern DNA replication across most organisms: (1) Double-stranded DNA replicates in a semiconservative manner, where the parental strands separate and serve as templates for the synthesis of new, complementary strands. (2) DNA replication in E. coli and other organisms is at least semidiscontinuous. One strand, often considered to replicate continuously in the direction of the replication fork's movement, may actually replicate discontinuously. The other strand replicates discontinuously, forming 1–2 kb Okazaki fragments in the opposite direction, allowing both strands to be synthesized in the 5'→3' direction. (3) DNA replication initiation requires a primer. In E. coli, Okazaki fragments are initiated with RNA primers that are 10–12 nucleotides long. (4) Most bacterial and eukaryotic DNAs replicate bidirectionally, though some, like ColE1, replicate unidirectionally.

Circular DNAs can replicate via the rolling circle mechanism, where one strand of the double-stranded DNA is nicked, and the 3'-end is extended using the intact strand as a template. This process displaces the 5'-end, and in phage λ, the displaced strand serves as a template for discontinuous, lagging strand synthesis.

Pol I is a highly versatile enzyme with three distinct activities: DNA polymerase, 3'→5' exonuclease, and 5'→3' exonuclease. The first two activities reside on a large domain of the enzyme, while the third is on a smaller, separate domain. The large domain, known as the Klenow fragment, can be isolated through mild protease treatment, yielding two protein fragments with all three activities intact. The structure of the Klenow fragment includes a wide cleft for DNA binding, with the polymerase active site located far from the 3'→5' exonuclease active site.

Among the three DNA polymerases in E. coli—Pol I, Pol II, and Pol III—only Pol III is essential for replication.