DNA replication is the process in which the parental DNA is copied into the identical two daughter DNA molecules. DNA replication is a basis for biological inheritance, which occurs in all living organisms. When the two strands of the DNA are separated, each strand can serve as the template for replicating a new complementary strand.
Replication in prokaryotes and eukaryotes occurs with the aid of different enzymes. The enzymes involved in DNA replication are helicases, DNA topoisomerase, primase, DNA polymerase, and ligase.
Helicase is the class of enzyme that separates the double strands of nucleic acids into single strands. It is also known as the helix destabilizing enzyme. Helicases require the energy which ATP provides. They are of two types, i.e., DNA helicase and RNA helicase. Helicase is involved in the different DNA modification processes like DNA replication, repair, recombination, transcription, translation, etc.
Helicase plays a crucial role in the DNA replication process. It is also known as Dna B protein. It is a ring-shaped hexamer containing six identical subunits. During the replication process, each of the single-stranded DNA helicases is loaded. Then the DNA replication occurs in the bi-direction, unwinding the strand and creating the V-shaped replication fork.
DNA topoisomerase is the enzyme that cuts and resolves the supercoils formed during the unwinding process. There are two types of supercoiling, positive and negative supercoiling.
A positive supercoil is twisted more and has many turns than negative supercoiling. It is faced towards the tightening of the coil, whereas the negative supercoil is faced towards losing the coils. Based on the twists and turns, topoisomerase acts on the strands. Topoisomerase forms the ester bond between a tyrosine residue of the enzyme and the DNA molecule. Then it reseals the cuts by forming a phosphodiester bond, and the enzyme gets released from the DNA.
Type I topoisomerase
Type I DNA topoisomerase cuts the negative supercoils and reseals them. It has nuclease and ligase activity only in the single strand of the double helix. To perform this activity, energy is not required.
Type II topoisomerase
Type II topoisomerase is also called DNA gyrase. It has both nuclease and ligase activity in both double helix strands. It relaxes both the negative and the positive supercoils. ATP is required as the energy to perform this activity.
Primase is a type of RNA polymerase that creates the RNA primer. Primase binds to the DNA helicase in bacteria in the form of primosomes. Helicase activates the primase then it synthesizes the primer. Primers are 4 to 15 nucleotides long. There are two different types of primase, i.e., Dna G and AEP. Dna G is in bacteria, and AEP is in eukaryotes and archaea.
Primase makes the short RNA primers on the single-stranded DNA template. These RNA primers are the short stretches of RNA complementary and antiparallel to the DNA template. It results in the formation of the hybrid duplex. The uracil (U) in RNA pairs with the adenine (A) in the DNA. Only one RNA sequence is required in the leading strand, while multiple primers are needed in the lagging strand. Then DNA polymerase adds the nucleotides to elongate DNA during the replication process.
DNA polymerases use deoxyribonucleotides and synthesize the DNA molecules by assembling the nucleotides. It matches the correct nucleotides and then joins the adjacent nucleotides to each other. DNA polymerase adds nucleotides only in the 3′ end of DNA. DNA polymerase is also involved in proofreading and error correction. DNA polymerase has a single active site from which it catalyzes the addition of any of the four deoxynucleoside triphosphates. DNA polymerase can add as many as 1000 nucleotides per second to the primer strand.
Prokaryotic DNA polymerase
The prokaryotic DNA polymerases are DNA polymerases I, II, III, IV, and V.
DNA polymerase I
DNA polymerase I or Pol I is encoded by the polA gene. In a single bacteria, about 400 Pol I molecules are present. Before being dissociated from the template strand, DNA polymerase I only make an average of 20 phospho-diester bonds. After the formation of the Okazaki fragment, RNA primers are removed from the lagging strand. Then DNA polymerase I adds the DNA nucleotides.
Pol I performs the four enzymatic activities.
- It performs the 5’-3′ DNA-Dependent DNA polymerase activity. It requires the 3′ primer site and a template strand.
- It performs the 3’-5′ exonuclease activity. It means the ability to remove the nucleotides from the 3′ end of the chain. It helps in the proofreading activity.
- It performs the 5’-3′ exonuclease activity. It removes the nucleotides from the 5′ end of DNA or from an RNA primer. It helps in DNA replication and repair.
- It performs 5’-3′ RNA-Dependent DNA polymerase activity.
DNA polymerase II
The pol B gene encodes DNA polymerase II or Pol II. Polymerase II does not have the 5’-3′ exonuclease activity.
DNA polymerase III
DNA polymerase III has proofreading activity and corrects DNA replication errors using the exonuclease activity working 3′ to 5′ direction. DNA polymerase III binds the RNA primer and keeps on elongating the DNA chain by adding the deoxynucleotides. It can add the nucleotides only in the 3′ hydroxyl end of the growing chain, due to which the synthesis of the daughter molecule occurs in the 5′ to 3′ direction.
DNA polymerase III enzymes comprise the ɑ, ε, and θ subunits.
These subunits perform their functions. The ɑ is encoded by the dnaE gene, and it has polymerase activity. The ε subunit is encoded by the dnaQ gene and has the3’-5′ exonuclease activity. The holE gene encodes the θ subunit. It stimulates the ε subunit’s proofreading activity.
DNA polymerase IV
DNA polymerase IV is encoded by the dinB gene. It is used in DNA repair.
DNA polymerase V
DNA polymerase V is encoded by the umuC and umuD genes during the SOS repair of DNA. SOS repair is cells’ last resort repair mechanism when exposed to a high level of mutagen or radiation.
|Name of enzymes||Function|
|Helicase||Unwinds the double strand|
|Primase||Synthesizes the RNA primer|
|DNA polymerase III||Add nucleotides and proofreading|
|DNA polymerase I||Removes RNA primer|
|DNA topoisomerase I||Cuts and reseals single strands|
|DNA topoisomerase II||Cuts and reseals both single and double strands|
|DNA ligase||Joins the fragments together|
Eukaryotic DNA polymerases
DNA replication in eukaryotes occurs in two different places, i.e., the nucleus and mitochondria. The eukaryotic DNA polymerases are α, β, γ, 𝛅 and ε
|Name of enzyme||Location||Size|
|DNA polymerase alpha (ɑ)||Nucleus||>250 kD|
|DNA polymerase beta (β)||Nucleus||36-38 kD|
|DNA polymerase gamma (γ)||Mitochondria||160-300 kD|
|DNA polymerase delta (δ)||Nucleus||170 kD|
|DNA polymerase epsilon (ε)||Nucleus||256 kD|
DNA polymerase alpha (ɑ)
Polymerase alpha initiates the synthesis of the strand on both the leading and lagging strand. DNA polymerase ɑ has primase activity, but the exonuclease activity is absent.
DNA polymerase beta (β)
DNA polymerase beta plays a crucial role in base excision repair (BER), essential for maintaining the DNA and in replication, recombination, etc.
DNA polymerase gamma (γ)
DNA polymerase gamma is crucial for mitochondrial DNA replication and repair.
DNA polymerase delta (δ)
DNA polymerase delta is involved in DNA replication and repair in eukaryotes.
DNA polymerase epsilon (ε)
DNA polymerase epsilon helps synthesize the leading strand of DNA and base excision repair.
In the lagging strands, different Okazaki fragments are formed. DNA ligase helps join the strands together by forming the phosphodiester bond. A phosphodiester bond is formed between the 3′ hydroxyl end and 5′ phosphate end of the two DNA strands. Making the phosphodiester bond requires a free OH group at the 3′ end and phosphate group at the 5′ end of the other DNA strand. To perform this action, energy is required, which is provided by the adenosine triphosphate (ATP). DNA ligase is used in DNA replication, repair, and recombination.
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