Introduction
In the course of DNA replication, a single strand of DNA duplicates itself several times. It is a type of biological polymerisation that occurs in three stages: initiation, elongation, and termination, in that order. It is a reaction that is catalysed by an enzyme. When it comes to DNA replication, DNA Polymerase is the most important enzyme. DNA replication in eukaryotes is a conserved system that only allows for DNA replication to occur once throughout each cell cycle. The replication of chromosomal DNA in eukaryotes is critical for the duplication of a cell and is required for the maintenance of the eukaryotic genome.
Steps in DNA Replication
- Initiation.
- Primer Synthesis.
- Leading Strand Synthesis.
- Lagging Strand Synthesis.
- Primer Removal.
- Ligation.
- Termination.
Initiation
A specific region of DNA known as the origin of replication, which has a precise sequence that can be identified by initiator proteins known as DnaA, is where DNA replication begins to take place. They bind to the DNA molecule at the origin sites, indicating that it is ready for the docking of other proteins and enzymes that are required for DNA replication to take place there. An enzyme known as helicase is recruited to the site of the helices in order to unwind them into single strands.
Helicases are enzymes that break hydrogen bonds between base pairs in a way that is energy dependent. The replication fork is a spot or region of DNA that is now recognised as such. Once the helices have been unravelled, a class of proteins known as single-strand binding proteins (SSB) bind to the unwound areas and prevent them from becoming annealed. As a result, the replication process is initiated, and the replication forks move in opposite directions along the DNA molecule to complete the process.
Primer Synthesis
Primer Synthesis known as DNA polymerases, these enzymes are responsible for the creation of a new, complementary strand of DNA from an existing strand of DNA by using the old strand as a template. Additionally, they play a crucial role in DNA repair and recombination, in addition to replicating DNA. DNA polymerases, on the other hand, are unable to initiate DNA synthesis on their own and require the presence of a 3′ hydroxyl group in order to begin the addition of complementary nucleotides. This is accomplished by the action of an enzyme known as DNA primase, which is a form of DNA-dependent RNA polymerase. It attaches a brief stretch of RNA to the existing DNA strands by synthesising it from scratch. This small section of DNA is referred to as a primer, and it contains 9-12 nucleotides. This provides DNA polymerase with the necessary platform for it to begin duplicating a DNA strand from the beginning. As soon as the primers have been generated on both DNA strands, DNA polymerases can be used to extend the primers to form new DNA strands, which is known as extension.
The unwinding of DNA may result in supercoiling in the areas that follow the fork in the DNA strand. This relaxation of the DNA supercoils is accomplished by a specific enzyme known as topoisomerase, which attaches to a DNA stretch just before the replication fork. It does this by creating a nick in the DNA strand, which allows the supercoil to be relieved.
Leading Strand Synthesis
In order to synthesise DNA in the 5′ 3′ direction, DNA polymerases can only add new nucleotides to the 3′ end of an existing strand, and hence they can only synthesise DNA in the 5′ 3′ direction. However, because the DNA strands run in opposite directions, the synthesis of DNA on a single strand can continue in a continuous fashion. The leading strand is the strand that comes before the other strands. In this case, DNA polymerase III (DNA pol III) identifies the 3′ OH end of the RNA primer and adds additional complementary nucleotides to the RNA primer strand. As the replication fork develops, additional nucleotides are continuously added to the existing strand, resulting in the formation of the new strand.
Lagging Strand Synthesis
When DNA is synthesised on the other strand, it occurs in a discontinuous fashion, with a sequence of short fragments of new DNA being generated in the 5′ 3′ direction. A collection of these pieces is referred to as an Okazaki fragment, and they are later combined to form a continuous chain of nucleotides. This strand is referred to as the lagging strand because the process of DNA synthesis on this strand occurs at a slower rate than on the other strands.
In this case, the primase adds primers in a number of locations along the unwinding strand. DNA polymerase III extends the primer by adding new nucleotides, and it terminates when it comes into contact with a previously produced fragment. Consequently, it must release the DNA strand and glide farther upstream in order to begin the extension of another RNA primer sequence. During the replication process, a sliding clamp holds the DNA in place as it moves from one location to another.
Primer Removal
Despite the fact that new DNA strands have been created, the RNA primers that were previously present on the newly generated strands must be replaced with DNA. Specifically, the enzyme DNA polymerase I is responsible for this activity (DNA pol I). Because of its 5′ 3′ exonuclease activity, it is able to precisely remove RNA primers from DNA, and the 5′ 3′ DNA polymerase activity is able to replace them with fresh deoxyribonucleotides.
Ligation
It is still possible to see gaps or nicks between neighbouring Okazaki fragments after the primer removal has been finished on the lagging strand. By forming a phosphodiester link between the 5′ phosphate and the 3′ hydroxyl groups of neighbouring fragments, the enzyme ligase recognises and seals these nicks in the DNA structure.
Termination
This replication mechanism comes to a halt at certain termination sites, each of which has a distinct nucleotide sequence. This sequence is recognised by specialist proteins known as tus, which bind to certain sites, preventing the helicase from proceeding through them. When the helicase comes into contact with the tus protein, it detaches from the strand along with the single-strand binding proteins that are adjacent.
Conclusion
In eukaryotes, the vast bulk of DNA synthesis takes place during the S phase of the cell cycle, and the entire genome must be unravelled and replicated in order to produce two daughter copies of the organism.
It takes three essential phases for DNA replication to occur: first, the opening of the double helix and separation of the DNA strands, followed by the priming of the template strand, and finally the construction of the new DNA segment. A specific site known as the origin of the DNA double helix uncoils during the process of separation of the two strands of DNA double helix. The consequence of DNA replication is the formation of two DNA molecules, each of which contains one new and one old nucleotide chain. This is why DNA replication is referred to as semi-conservative; half of the chain is derived from the original DNA molecule, while the other half is completely different.