Initiation of DNA Replication:
DNA Replication-Initiation stage
DNA Replication-Initiation stage
The E.Coli replication origin, called “Ori.C”, consists of 245 base pairs, many of which are highly conserved among bacteria. The key sequences for this discussion are two series of short repeats; there repeats of a 13 base pair sequence and four repeats of a 9 base pair sequence.
At least “Eight different enzymes” (or) “Proteins” participate in the initiation phase of replication. They open the DNA helix at the origin and establish a preparing complex that sets the stage for subsequent reactions.
The key component in the initiation process is the Dna.A protein. A complex of about twenty Dna.A protein molecule binds to the four Nine bp repeats in the origin. In a reaction that requires ATP and is facilitated by the bacterial histone like protein “HU”, the Dna.A protein recognizes and successfully denatures the DNA in the region of the there 13 base pairs repeats, which are rich in A=T pair.
The Dna.B protein then binds to this region in a reaction that requires the Dna.C protein. Dna.B is a “Helicase” that unwinds duplex DNA. The helicases constitute a class of enzymes that can move along a DNA duplex utilizing a class of enzymes that can move along a DNA duplex utilizing the energy of ATP hydrolysis to separate the strands. The separated strands are inhibited from subsequently re-annealing by the E.Coli Single Stranded Binding protein (SSBP), which binds to both separated strands. Multiple molecules of SSB bind cooperatively to single – stranded DNA, stabilizing the separated DNA strands and preventing re-naturation.
DNA replication must be occurs only once in each cell cycle. Initiation is the only phase of replication that is regulated, but the mechanism is not yet well understood.
The Dna.A protein hydrolyses its tightly bound ATP slowly (about 1 hour) to form an inactive Dna.A – ADP complex. Reactivating this complex is facilitated by an interaction between Dna.A protein and acidic phospholipids in the bacterial plasma membrane. Initiation at in appropriate times is prevented by the presence of the inactive Dna.A-ADP complex by the binding of a protein called “Ici.A Inhibitor of chromosomal initiation) to the 13 base pair repeats, and perhaps by other factors.