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Initiation

All cells must finish DNA replication before they can proceed for cell division. Media conditions that support fast growth in bacteria also couples with shorter inter-initiation time in them, i.e. the doubling time in fast growing cells is less as compared to the slow growth. In other words, it is possible that in fast growth conditions the grandmother cells starts replicating its DNA for grand daughter cell. For the same reason, the initiation of DNA replication is highly regulated. Bacterial origins regulate orisome assembly, a nuclei-protein complex assembled on the origin responsible for unwinding the origin and loading all the replication machinery. In E. coli, the direction for orisome assembly are built into a short stretch of nucleotide sequence called as origin of replication (oriC) which contains multiple binding sites for the initiator protein DnaA (a highly homologous protein amongst bacterial kingdom). DnaA has four domains with each domain responsible for a specific task. There are 11 DnaA binding sites/boxes on the E. coli origin of replication out of which three boxes R1, R2 and R4 (which have a highly conserved 9 bp consensus sequence 5' - TTATC/ACACA) are high affinity DnaA boxes. They bind to DnaA-ADP and DnaA-ATP with equal affinities and are bound by DnaA throughout most of the cell cycle and forms a scaffold on which rest of the orisome assembles. The rest eight DnaA boxes are low affinity sites that preferentially bind to DnaA-ATP. During initiation, DnaA bound to high affinity DnaA box R4 donates additional DnaA to the adjacent low affinity site and progressively fill all the low affinity DnaA boxes. Filling of the sites changes origin conformation from its native state. It is hypothesized that DNA stretching by DnaA bound to the origin promotes strand separation which allows more DnaA to bind to the unwound region. The DnaC helicase loader then interacts with the DnaA bound to the single-stranded DNA to recruit the DnaB helicase, which will continue to unwind the DNA as the DnaG primase lays down an RNA primer and DNA Polymerase III holoenzyme begins elongation.