Initiation of DNA Replication

DNA replication is initiated specifically from an origin. Initiation appears to be the major target for the control of replication. Two requirements for replication initiation are as follows:

> 1. A nucleotide sequence that specifically binds initiation proteins, and
> 2. A mechanism that generates a primer terminus for DNA polymerase to extend.

The two most straightforward ways to generate a primer terminus at the origin are as follows:

> 1. Nicking a strand of the parental duplex to expose a 3' hydroxyl terminus
> 2. Synthesizing an RNA primer to expose a 3' hydroxyl ribonucleotide terminus.

E. coli - The DNA replication origin of E. coli is called oric. The sequence is 245 base pairs long, with four repeats of a 9-base-pair sequence that binds to an initiation protein (the dnaA gene product). To the left of these sites, depicted in Figure 24.37, are three direct repeats of a 13-base-pair sequence that is rich in A and T and is thus denatured relatively easily. The sequence also contains binding sites for several basic proteins (HU and IHF) that facilitate DNA bending, an important step in the sequence leading to initiation. Steps in the process are as follows:

> 1. Binding of 10-20 molecules of a DnaA/ATP complex. This causes the DNA to bend. Bending the DNA unwinds the 13-base pair regions. DnaA is activated for this step by reacting with a phospholipid called cardiolipin.

> 2. The DnaC/DnaB protein complex binds to both forks of the opened loop. The helicase (DnaB) acts to open the structure further.

> 3. DnaG (primase) binds and synthesizes RNA primers. RNA polymerase may also synthesize some primers.

> 4. DNA polymerase III holoenzyme extends both leading and lagging strands.

Mitochondria - Figure 24.39 illustrates the unusual DNA replication initiation scheme employed by animal cell mitochondria. This involves two unidirectional replication processes.

> 1. Initiation occurs at a fixed origin on the L strand.

> 2. The heavy strand is displaced by replication on the L strand

> 3. About 2/3 of the way around the circle, a second origin is exposed on the displaced H strand.

> 4. Unidirectional synthesis from the second origin proceeds back in the other direction.

The mitochondrial system, with its two unidirectional modes, avoids the need to simultaneously synthesize leading and lagging strands. As a result, both strands are made in a continuous fashion--no Okazaki fragments.

See also: Replication of Linear Genomes

INTERNET LINK: Regulation of Eukaryotic DNA Replication