Once oriC had been cloned it became possible to study the initiation of DNA replication in vitro. Arthur Kornberg and coworkers performed the difficult but rewarding task of purifying the enzymes that participate in the initiation process and elucidating their function. The first two stages in the replication initiation process, origin recognition and DNA unwinding, were described above. During the next stage, helicase loading, the DnaB helicase is loaded onto each of the single strands in the unwound AT-rich region of oriC. Both DnaC and DnaA·ATP participate in loading the DnaB hexamers. ATP hydrolysis leads to DnaC release, freeing each DnaB helicase to move in a 5'→3'direction and thereby extend the unwound region of oliC to about 65 nucleotides.
Next, two DnaG (primase) molecules enter the complex one primase is associated with each DnaB helicase. Each primase then synthesizes a leading strand primer. Formation of these RNA primers ensures that replication begins exclusively at oriC and that a pair of replication forks is formed for bidirectional replication. primase has a zinc binding domain at its N-terminus, a Dna8 binding domain at its C-terminus, and an RNA polymerase domain in the middle. The final stage of the initiation cycle involves the addition of the sliding clamp and DNA polymerase Ⅲ holoenzyme (see below) with the concomitant release of DnaA · ADP from the DnaA boxes.