DNA replication in prokaryotes is the process of copying of prokaryote genome the same as that of its original genome that is passed on to the daughter cells.
- Replication of DNA in prokaryotes has been well studied because of its small genome size and having a single specific origin of replication.
- The chromosome of a prokaryotic organism is circular with a less extensive coiling structure than that of a eukaryotic chromosome which is highly coiled around the proteins.
- E. coli is the most studied prokaryote while other prokaryote shows similar types of replication.
- In a single circular chromosome, E. coli contains 4.6 million base pairs which get replicated in approximately 42 minutes i.e, 1000 nucleotides are added in one second.
- DNA replication is a bi-directional process that means two individual strand proceeds in the different direction being a lagging strand and a leading strand.
- Replication in prokaryote occurs in the cell cytoplasm.
- Various numbers of proteins and enzymes are involved during the replication process each having their own specific role.
- To begin the replication process, a specific nucleotide sequence called the origin of replication must be known. As in E. coli, it has a specific single origin of replication on its one chromosome which is known as Ori C. Most prokaryotes also contain the Ori C site of replication origin.
Structure of Ori C
- In E. coli, Ori C is the specific site at which the DNA starts its replication.
- Abbreviation of Ori C is the Col E. chromosome and is located at the 84.5 mpu position of the E. coli genome opposite of the termination site.
- Two distinct regions are present in the Ori C site, one is called 9 mer that has 9 nucleotides and the other is 13 mer that has 3 nucleotides. Both these are repeating sequences where 13 mer is rich in A-T bases.
- The 9 mer is also referred to as the DNA-A box and binds at the site of DNA-A protein that is when the replication starts.
In prokaryotes, DNA replication completes in three stages:
- The DNA-A protein recognizes the Ori C site and binds to the DNA-A box which is a 9 mer.
- Then, the 9 mer gets wrapped around the DNA-A protein which melts the 13 mer base pair A-T rich segment.
- ATP is required for the DNA-A protein as it is ATP dependent therefore, ATP hydrolysis occurs in this process where DNA-A protein recruits Dna-B protein.
- DNA-B protein has a helicase activity that unwinds the strands of DNA by breaking the hydrogen bond between them forming a replication fork.
- Another protein called single-strand binding protein (SSB) binds each strand of DNA to prevent the strands from winding back.
- Topoisomerase II or DNA gyrase enzyme gets involved to release the tension caused by the supercoiling.
- Another enzyme RNA primase synthesizes the RNA primer and complements to the DNA.
- Then DNA polymerase III adds the nucleotide one by one complementing to the template strand.
- The addition of nucleotides by DNA polymerase III enzymes is 1000 nucleotides per second. Energy is required for this process which can be obtained from the nucleotide with three phosphates attached to them.
- One DNA strand complementary to 3’ to 5’ is synthesized continuously towards the replication fork and is known as the leading strand while the other complementary to 3’ to 5’ extends away from the replication fork in a discontinuous manner with Okazaki fragments and is called lagging strand.
- One primer is enough for the leading strand while the lagging strand requires a new primer for each Okazaki fragments.
- The nicks that remain between the newly synthesized DNA are sealed by the DNA ligase enzyme.
- At a certain specific points, the replication stops and starts terminating.
- That specific point is known as the termination site (Ter site) where it gets bound by the Tus protein (Termination utilization protein).
- The formed Ter – Tus complex forms a trap for replication fork and cause termination which occurs at the Ter sequence on the genome of E. coli.
- This Ter sequence is where Tus protein binds and forms Ter – Tus complex.
- Ter sequence contains 10 different sequences from Ter A to Ter J in which five Ter sequences from Ter A to E move towards left and another 5 Ter from F to J sequences move towards the right.
- When two replication fork moves toward the opposite direction and at the same time Topoisomerase II unlinks the two opposite moving strands by breaking the phosphodiester bond between two nucleotides.
- Finally, after the separation of two individual daughter strands, the DNA ligase enzyme seals the gap between the two DNA fragments and forms the phosphodiester bond.
- Hence, two individual daughters DNA are formed with each containing one duplicate parent DNA complementing to its own newly formed DNA.