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Initiation

The initiation of DNA replication is mediated by DnaA, a protein that binds to a region of the origin known as the DnaA box. In E. coli, there are 4 DnaA boxes, each of which contains a highly conserved 9 bp consensus sequence 5' - TTATCCACA - 3'. Binding of DnaA to this region causes it to become negatively supercoiled. Following this, a region of OriC upstream of the DnaA boxes (known as DnaB boxes) become melted. There are three of these regions, and each is 13 bp long, and AT-rich (which facilitates melting because less energy is required to break the two hydrogen bonds that form between A and T nucleotides). This region has the consensus sequence 5' - GATCTNTTNTTTT - 3. Melting of the DnaB boxes requires ATP (which is hydrolyzed by DnaA). Following melting, DnaA recruits a hexameric helicase (six DnaB proteins) to opposite ends of the melted DNA. This is where the replication fork will form. Recruitment of helicase requires six DnaC proteins, each of which is attached to one subunit of helicase. Once this complex is formed, an additional four DnaA proteins bind to the original four DnaA proteins to form four DnaA dimers. DnaC is then released, and the prepriming complex is complete. In order for DNA replication to continue, SSB protein is needed to prevent the single strands of DNA from forming any secondary structures and to prevent them from reannealing, and DNA gyrase is needed to relieve the stress (by creating negative supercoils) created by the action of DnaB helicase. The unwinding of DNA by DnaB helicase allows for primase (DnaG) and RNA polymerase to prime each DNA template so that DNA synthesis can initiate.