The initial replication fork requires the separation of the two strands of the DNA duplex to provide a template for the synthesis of both the RNA primer and new DNA. Specific genomic DNA sequence direct the initiation of DNA Replication. The specific sites at which DNA unwinding and initiation of replication occur are called Origin of Replication.
The Replicon Model of Replication Initiation:
In 963 Frabcois Jacob, Sydney Brenner and Jacques Cuzin proposed a model to explain the events controlling the initiation of replication in bacteria. It proposed two components that control the initiation of replication :
- Replicator: Replicator is defined as the entire set of cis-acting DNA sequences that is sufficient to direct the initiation of Replication.
- Initiator: Initiator is the protein which specifically recognizes a DNA elements in the replicator and activates the initiation of replication. The initiator protein is the only sequence specific DNA-binding protein involved in the initiation of replication.
The model proposed that all the DNA replicated from a particular origin as a replicon. In prokaryotes since there is only one origin of replication at the single chromosome, the entire chromosome is a single replicon. In contrast the presence of multiple origins of replication divides each eukaryotic chromosomes into multiple replicons.
The origin of replication is the site on DNA where the DNA is unwound and DNA synthesis initiates. Although the origin of replication is always part of the replicator, sometimes the origin of replication is only a fraction of the replicator.
DNA Sequences of Replicator Share two common features:
- First, they include a binding site for the initiator protein.
- They include a stretch of AT-rich DNA that unwinds readily but not spontaneously. Unwinding of DNA at replicators is controlled by the replication Initiation Proteins.
The single replicator required for E-Coli chromosomal replication is called oriC. There are two repeated motif that are critical for oriC function. The 9-mer motif is the binding site for the E-Coli initiator, DnaA, and is repeated four times at oriC. The 13-mer motif, repeated 3 times, is the initial site of ssDNA formation during initiation.
Initiator proteins typically perform 3 different functions:
- Binding to the replicator – Bind a specific sequence DNA sequence within the replicator.
- Unwind DNA – once bound to the DNA, they frequently distort or unwind a region of DNA adjacent to the site of binding.
- Recruiting other replication proteins – Initiator proteins interact with additional factors required for replication initiation, thus recruiting them to the replicator.
In Prokaryotes, for example, the E-coli initiator, DnaA binds the repeated 9-mer elements in oriC and is regulated by ATP. When bound to ATP, DnaA also interacts with DNA in the region of the repeated 13-mer repeats of oriC. These additional interactions result in the separation of the DNA strands over more than 20bp within the 13-mer repeat region. this unwound DNA provides an ssDNA template for additional replication proteins such ad DNA helicase to begin the RNA and DNA synthesis steps of replication.
In Eukaryotes, the initiator is known as the origin recognition complex (ORC). It is a complex formed of 6 proteins. Like DnaA in E-Coli, ORC binds and hydrolyze ATP. ATP is required for sequence-specific DNA binding at the origin. Unlike DNA binding ORC at the replicator does not separate the DNA strands. ORC is , however, required to recruit all the remaining replication proteins to the replicator before it unwinds. Unwinding the strands requires the origin to get activated. Origin activation occurs only when the cell enters S phase of cell cycle.
Unlike prokaryotic initiator, ORC of eukaryotes have two functions:
- Binding to the replicator
- Recruiting other replication proteins
once the initiator binds to the replicator, the remaining steps in the initiation of replication is largely driven by the protein-protein interactions and protein-DNA interactions that are sequence independent. The end result is the assembly of two replication fork.