DNA Replication

Replication of DNA

DNA replication is the process by which DNA makes an exact copy of itself during cell division. This ensures that each daughter cell receives an identical copy of the genetic material.

Steps in DNA replication

1. Initiation of DNA Replication

The primary step for the double helical DNA molecule to be replicated is the separation of the two strands of the parent DNA molecule. The separation begins at the small region of the DNA molecule known as the origin of replication. In E. Coli, the origin of replication is called “oriC,” and in eukaryotes, it is referred to as the consensus sequence. The single chromosome found in E. Coli has only one origin of replication, whereas the eukaryotic chromosome has multiple origins of replication.

2. Formation of replication fork

At the origin of replication, the two strands begin to unwind or separate. It assumes the V or Y-shaped structure called as replication fork. The separated strand in the replication fork acts as a template for the synthesis of new DNA.

 Requirements for DNA strand separation:

Dna A proteins

Dna B proteins (Helicase)

Single-stranded DNA binding proteins

Topoisomerases

a. Dna A proteins: This is the key component for the initiation of DNA replication. There are 20-50 monomers of the Dna A protein that bind to a specific nucleotide sequence at the origin of replication.

b. Dna B protein (Dna Helicase): After the binding of Dna A protein at the origin of replication, the next protein, Dna helicase, binds to the unwound region and catalyzes the separation of DNA. Helicases are responsible for unwinding the double helix, using energy from ATP. They move along the DNA helix and separate the strands like a zipper.

c. Single-stranded DNA-binding proteins: These enzymes are also known as DNA helix-destabilizing proteins. They bind only to single-stranded DNA, keep the two strands apart, and provide a template for new DNA synthesis. It is also believed that the SSB protein protects single-strand DNA from degradation by nucleases.

d. Topoisomerases (Super helix relaxing proteins): Rapid unwinding of the DNA strand can cause tension and lead to supercoiling of the helix, similar to how rapid separation of a rope's strands can create knots or coils. This tension is relieved by the action of topoisomerase. This enzyme has nicking (cutting) and closing activities: it introduces nicks or breaks in the non-replicating region of DNA, allowing one strand to rotate around the other and relieve tension. The break is then sealed by ligation, permitting the replication fork to continue.

There are two types of Topoisomerases:

Type I – Topoisomerase: It cuts the single DNA strand to overcome the problem of supercoils and reseals the strand again.

Type II – Topoisomerase (DNA gyrase): It is also known as DNA gyrase, which cuts both strands and reseals them to overcome the problem of supercoiling.

3. DNA Replication / Elongation

Once the replication fork is formed, the process of elongation begins, where the new complementary DNA strands are synthesized. DNA polymerase cannot initiate the synthesis of a new strand on its own; it requires a free 3’-OH group to add nucleotides.

A specialized RNA polymerase called primase synthesizes a short RNA primer (typically 5–50 nucleotides long, species-dependent), which is complementary and antiparallel to the DNA template. This RNA primer provides the free 3’-OH group necessary for DNA polymerase to begin elongation. Once the primer is in place, DNA polymerase III adds deoxyribonucleotides to the 3’ end of the primer in a 5’ → 3’ direction, using the template strand read in the 3’ → 5’ direction.

Reaction Equation

dNTP + (dNMP)n → (dNMP)n+1 + PPi (pyrophosphate)

Where, dNTP = deoxyribonucleoside triphosphate (dATP, dGTP, dCTP, dTTP)
DNA polymerase catalyzes the addition of nucleotides
PPi = pyrophosphate (a by-product)

Pyrophosphatase rapidly hydrolyzes pyrophosphate (PPi) into two inorganic phosphate molecules (2Pi): PPi → 2Pi

This hydrolysis releases free energy that drives the polymerization reaction forward, ensuring the process is energetically favorable.

The synthesis of two new DNA strands simultaneously takes place in the opposite direction. One is in a direction 5’à3’ towards the replication fork, which is continuous; such a strand is called as leading strand. The other in a direction 5’à 3’ away from the replication fork, which is discontinuous by forming small fragments, such strand, is called as the lagging strand.

Thus, DNA strands are antiparallel, and replication occurs bidirectionally at each replication fork. Therefore, two new strands are synthesized differently:

🔸 Leading Strand
Synthesized continuously in the same direction as the replication fork movement (5’ → 3’).
Requires only one RNA primer at the origin.

🔸 Lagging Strand
Synthesized discontinuously in the opposite direction of the replication fork.
Short stretches of DNA, called Okazaki fragments, are synthesized in 5’ → 3’ direction.
Each fragment requires a new RNA primer.
Later, these fragments are processed and joined to form a continuous strand.

4. Excision of RNA Primers

DNA polymerase I removes RNA primers by its 5’ → 3’ exonuclease activity.
It also fills in the gap with the correct DNA nucleotides using its polymerase activity.

5. Joining of DNA Fragments

 
DNA ligase seals the nicks between adjacent Okazaki fragments by forming phosphodiester bonds between the 3’-OH and 5’-phosphate ends.
Ligase Reaction in E. coli

E. coli DNA ligase uses NAD+ as an energy source (in contrast to eukaryotic ligases, which use ATP):
DNA ligase + NAD+ → Ligase–AMP + NMN (Nicotinamide mononucleotide)
Ligase–AMP + DNA (nick) → Sealed phosphodiester bond + AMP + Ligase

6. Termination of Replication

In prokaryotes (e.g., E. coli), replication terminates when the two replication forks meet at the termination sites (ter sites) located opposite the origin (oriC). Tus protein (Termination utilization substance) binds specifically to these ter sites, forming a Tus–Ter complex. This complex halts the replication fork, preventing over-replication and ensuring proper completion of the replication process.