Steps involved in DNA Replication in Prokaryotes (E.coli)

In prokaryotes, the DNA is circular. Replication starts at a single origin (ori C) and is bi-directional and semi-conservative.

The region of replicating DNA associated with the single origin is called a replication bubble or replication eye and consists of two replication forks moving in opposite direction around the DNA circle.

During DNA replication, the two parental strands separate and each acts as a template to direct the enzyme catalysed synthesis of a new complementary daughter strand following the normal base pairing rule.

Three basic steps involved in DNA replication are Initiation, elongation and termination.

I. Initiation

Step 1: Binding of DNA around an initiator protein complex DNA-A ATP ~30-40.

The DNA B or helicase unwinds ori C (origin of replication) and extends the single stranded region for copying.

Step 2: Single strand binding protein (SSB) binds to this single stranded region to protect it from breakage and to prevent it from renaturing.

As the parental DNA is unwound by DNA helicases and SSB (travels in 5’-3’ direction), the resulting positive supercoiling (torsional stress) is relieved by topoisomerse I and II (DNA gyrase) by inducing transient single stranded breaks.

Step 3: The enzyme DNA primase (primase, an RNA polymerase) then attaches to the DNA and synthesises a short RNA primer to initiate synthesis of the leading strand of the first replication fork.

II. Elongation

Step 4: DNA polymerase III extends the RNA primer made by primase.

DNA polymerase possesses separate catalytic sites for polymerisation and degradation of nucleic acid strands.

All DNA polymerases make DNA in 5’-3’ direction

Leading strand synthesis

On the template strand with 3’-5’ orientation, new DNA is made continuously in 5’-3’ direction towards the replication fork. The new strand that is continuously synthesized in 5’-3’ direction is the leading strand.

Lagging strand synthesis

On the template strand with 5’-3’ orientation, multiple primers are synthesized at specific sites by primase (primosome complex) and DNA pol III synthesizes short pieces of new DNA (about 1000 nucleotides long) new DNA is in 5’-3’ direction.

These small DNA fragments that are discontinuously synthesises are called Okazaki fragments (named after the discoverer Reigi Okazaki). The new strand which is discontinuously synthesised in small fragments is called the lagging strand.

DNA polymerase III synthesizes DNA for both leading and lagging strands.

Step 5: After DNA synthesis by DNA pol III, DNA polymerase I uses its 5’-3’ exonuclease activity to remove the RNA primer and fills the gaps with new DNA.

Step 6: Finally DNA ligase joins the ends of the DNA fragments together.

III. Termination

Step 7: The two replication forks meet ~ 180 degree opposite to ori C, as DNA is circular in prokaryotes. Around this region there are several terminator sites which arrest the movement of forks by binding to the tus gene product, an inhibitor of helicase (Dna B).

Step 8: Once replication is complete, the two double stranded circular DNA molecules (daughter strands) remain interlinked. Topoisomerase II makes double stranded cuts to unlink these molecules.