DNA replication is a fundamental biological process by which a cell duplicates its entire DNA. DNA is a self-replicating structure, and the replication is catalysed by enzymes. Through DNA Replication, genetic information is passed on from one generation of cells to the next during cell division. It takes place in the nucleus of eukaryotic cells and the cytoplasm of prokaryotic cells.
The replication of DNA occurs during the synthesis phase, or S phase, of the cell cycle, before the cell enters mitosis or meiosis. In this process, initially, an enzyme called DNA helicase unwinds the DNA molecule, leading to the separation of its strands, and enzymes known as polymerases catalyse the formation of new DNA strands. The initiation of new DNA strands occurs with the help of a small RNA primer.
Steps of DNA Replication
The important steps involved in DNA replication are as follows:
Initiation of DNA Replication
- The replication process starts at specific sites on the DNA molecule called origins of replication.
- Enzymes, known as helicases, unwind and separate the DNA strands, creating a DNA Replication fork.
- An RNA primer is synthesised by an enzyme called primase.
- This primer provides a starting point for the DNA polymerase to attach nucleotides.
Elongation of DNA Replication
- DNA polymerase adds complementary nucleotides to the template strand.
- DNA polymerase only adds nucleotides in one direction, that is, the 5' to 3' direction, creating the new DNA strand in a 3' to 5' direction.
- The leading strand is synthesised continuously, following the replication fork movement.
- The lagging strand is synthesised discontinuously in small fragments called Okazaki fragments.
- DNA polymerase adds nucleotides to each fragment, with the help of RNA primers.
- After Okazaki fragments are synthesised, DNA polymerase replaces RNA primers with DNA and seals the gaps between fragments using DNA ligase.
Termination of DNA Replication
- Replication continues bidirectionally until both replication forks meet, completing the synthesis of the entire DNA molecule.
- The entire process is semi-conservative as each of the two copies consists of an original strand paired with a newly synthesised strand.
Role of Enzymes in DNA Replication
DNA is made up of a double helix of two complementary strands. Different enzymes are involved in various stages of replication, contributing to the unwinding of the DNA double helix, synthesis of new strands, and error correction. Here are some key enzymes and their roles in DNA replication:
1. DNA Helicase prokaryotes/Eukaryotes
- DNA helicase was discovered in E.coli in 1976. It is also called helix destabilising enzyme or unzipping or unwinding enzyme, since it separates the two strands of DNA at replication.
- They are the motor proteins that move directionally along the nucleic acid.
- It unwinds the DNA double helix by breaking the hydrogen bonds between complementary base pairs, creating the replication fork.
2. DNA Polymerase
DNA polymerases are the enzymes that synthesise the DNA molecules from ribonucleotides, the building blocks of DNA strands. The DNA polymerase reads the existing DNA strand to create the new strands that match the existing one, and also performs proofreading and error correction. The  DNA polymerase catalyses the extension of the 3' end of the DNA strand by one nucleotide at a time.
DNA polymerases can be further divided into two different families, which are as follows.
Prokaryotic DNA Polymerase Types and Functions
- DNA Polymerase I is coded by the polA gene. It is a single polypeptide and plays a part in recombination and fixing. It has both 5' and 3' exonuclease action. DNA polymerase I eliminates the RNA primer from the lagging strand by 5' exonuclease action and fills the hole.
- DNA Polymerase II is coded by the polB gene. It is comprised of 7 subunits. Its primary job is to repair and further reinforce DNA polymerase III. It has 3'exonuclease action.
- DNA Polymerase III is the primary enzyme for replication in E.coli. It is coded by the polC gene. The polymerisation and processivity rate is the most extreme in DNA polymerase III. It additionally has editing 3' exonuclease activity
- DNA Polymerase IV is coded by the dinB gene. Its principal job is in DNA repair during the SOS reaction when DNA replication is slowed down at the replication fork. DNA polymerases II, IV and V are translation polymerases.
- DNA Polymerase V is likewise engaged with translation synthesis during the SOS reaction and DNA repair.
Eukaryotic DNA Polymerase Types and Functions
- DNA polymerase α - It is the principal protein for replication in eukaryotes. It likewise has 3'????' exonuclease action for proofreading.
- DNA polymerase γ - The principal capability of DNA polymerase γ is to synthesise primers. The smaller subunit has a primase activity. The biggest subunit has polymerisation action. It frames a primer for Okazaki sections, which are then stretched out by DNA polymerase γ.
- DNA polymerase δ - The fundamental capability is DNA repair. It eliminates primers for Okazaki parts from the lagging strand.
- DNA polymerase ε - It is the super replicative enzyme for mitochondrial DNAÂ
3. Topoisomerase
- Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA.
- DNA topoisomerases prevent and correct types of topological problems.
- They do this by binding to DNA and cutting the sugar-phosphate backbone of either one (type I topoisomerases) or both (type II topoisomerases) of the DNA strands.
- This transient break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed.
- Since the overall chemical composition and connectivity of the DNA do not change, the DNA substrate and product are chemical isomers, differing only in their topology.
4. DNA Ligase
- DNA ligases play an essential role in maintaining genomic integrity by joining breaks in the phosphodiester backbone of DNA that occur during replication and recombination and as a consequence of DNA damage and its repair.
- Three human genes, LIG1, LIG3 and LIG4, encode ATP-dependent DNA ligases.
- DNA ligase seals the gaps between Okazaki fragments on the lagging strand by forming phosphodiester bonds.
- It creates a continuous DNA strand.
5. Primase
- DNA primase is an enzyme whose continual activity is required at the DNA replication fork.
- They catalyse the synthesis of short RNA molecules used as primers for DNA polymerases.
- These primers provide starting points for DNA synthesis by DNA polymerase.
- Primers are synthesised from ribonucleoside triphosphates and are four to fifteen nucleotides long.
6. Endonucleases
- These enzymes catalyse the cleavage of phosphodiester bonds within a DNA or RNA molecule.
- Unlike exonucleases, which remove nucleotides from the ends of DNA or RNA strands, endonucleases cleave within the molecule itself.
7. Single-Strand Binding Proteins
- Single-Strand Binding Proteins are DNA-binding proteins that attach to single-stranded DNA (ssDNA) during DNA replication.
- These proteins stabilise single-stranded DNA regions, preventing them from re-forming double-stranded structures.
Difference Between Prokaryotic and Eukaryotic Replication
The following points highlight the key differences between prokaryotic and eukaryotic DNA replication:
| Features | Prokaryotic Replication | Eukaryotic Replication |
|---|---|---|
Location | Cytoplasm | Nucleus |
Timing | Anytime before cell division | S-Phase of the cell cycle |
Rate | Faster (2000 bp/s) | Slower (100 bp/s) |
Enzymes | DNA gyrase, DNA Polymerase III | Helicase, Topoisomerase, DNA Polymerases α, β, ε, γ |
Replicons | One | Multiple |
Ori Sites | One | Multiple |
Terminus | One | Multiple |
Okazaki Fragments | Longer (1000-2000 Nucleotides) | Shorter (100-200 Nucleotides) |
Nucleus | Absence of a membrane-bound nucleus | Presence of a membrane-bound nucleus |
Accuracy | More accurate | Less accurate geneticc Material |
Initiation Point | Typically has a single origin of replication | Multiple origins of replication along chromosomes |
Complexity | Simple and less complex | Complex and involves various stages of mitosis |
Importance of DNA Replication
The importance of DNA Replication is as follows:
- DNA replication helps in the transfer of genetic information from one generation to the next during cell division, which enables the inheritance of traits and characteristics and maintains genetic diversity.
- DNA replication is important for repairing damaged DNA.
- DNA replication is essential for cell division, which provides each new daughter cell with a complete set of genetic instructions that is required for its proper functioning and development.
- Mutations and genetic variations, which arise during replication, contribute to the diversity of species over generations and can lead to the emergence of new traits.
- Accurate DNA replication helps prevent mutations that can lead to genetic disorders and diseases.