Mechanism of Initiation of Replication
At least 10 different enzymes or proteins participate in the initiation phase of replication. They open the DNA helix at the origin and establish a prepriming complex for subsequent reactions. The crucial component in the initiation process is the DnaA protein, a member of the AAA+ ATPase protein family (ATPases associated with diverse cellular activities). Many AAA+ ATPases, including DnaA, form oligomers and hydrolyze ATP relatively slowly. This ATP hydrolysis acts as a switch mediating interconversion of the protein between two states. In the case of DnaA, the ATP-bound form is active and the ADP-bound form is inactive.
Eight DnaA protein molecules, all in the ATP-bound state, assemble to form a helical complex encompassing the R and I sites in oriC. DnaA has a higher affinity for the R sites than I sites, and binds R sites equally well in its ATP or ADP-bound form. The I sites, which bind only the ATP-bound DnaA, allow discrimination between the active and inactive forms of DnaA. The tight right-handed wrapping of the DNA around this complex introduces an effective positive supercoil. The associated strain in the nearby DNA leads to denaturation in the A:T-rich 'DUE' (DNA Unwinding Element)region. The complex formed at the replication origin also includes several DNA-binding proteins- Hu, IHF and FIS that facilitate DNA bending.
The DnaC protein, another AAA+ ATPase, then loads the DnaB protein onto the separated DNA strands in the denatured region. A hexamer of DnaC, each subunit bound to ATP, forms a tight complex with the hexameric, ring-shaped DnaB helicase. This DnaC-DnaB interaction opens the DnaB ring, the process being aided by a further interaction between DnaB and DnaA. Two of the ring-shaped DnaB hexamers are loaded in the DUE, one onto each DNA strand. The ATP bound to DnaC is hydrolyzed, releasing the DnaC and leaving the DnaB bound to the DNA.
Loading of the DnaB helicase is the key step in replication initiation. As a replicative helicase, DnaB migrates along the single-stranded DNA in the 5'→3' direction, unwinding the DNA as it travels. The DnaB helicases loaded onto the two DNA strands thus travel in opposite directions, creating two potential replication forks. All other proteins at the replication fork are linked directly or indirectly to DnaB.
Read more about this topic: DnaB Helicase
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