DNA Replication

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 catalyzed 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.

DNA Replication 

DNA, or Deoxyribonucleic acid, is the genetic material in the majority of living organisms. The process of DNA replication is a biological mechanism that generates two identical copies of DNA from an original strand. 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 strand, 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.

DNA Replication Steps

The important steps involved in DNA replication are as follows:

Initiation of DNA Replication

The replication process starts at a 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 synthesized by an enzyme called primase. This primer provides a starting point for DNA polymerase to attach nucleotides.

DNA Polymerization (Elongation)

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 synthesized continuously, following the replication fork movement. The lagging strand is synthesized discontinuously in small fragments called Okazaki fragments. DNA polymerase adds nucleotides to each fragment, with the help of RNA primers. After Okazaki fragments are synthesized, DNA polymerase replaces RNA primers with DNA and seals the gaps between fragments using DNA ligase.

Termination of DNA Replication

Replication continues bi directionally 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 synthesized strand.

Role of Enzymes in DNA Replication

DNA is made up of a double helix of two complementary stands. 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:

DNA Helicase prokaryotes/Eukaryotes

DNA helicase was discovered in E.coli in 1976. It is also called helix destabilize 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 unwind the DNA double helix by breaking the hydrogen bonds between complementary base pairs, creating the replication fork.

DNA Polymerase

DNA polymerases are the enzymes that synthesize 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 the proofreading and error correction. The  DNA polymerase catalyzes the  DNA template and directs the extension of the 3' end of the DNA strand by 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 furthermore 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 furthermore a reinforcement of DNA polymerase III. It has 3'????' exonuclease action.
•  DNA Polymerase III is the primary enzyme for replication in E.coli. It is coded by polC gene. The polymerization 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 fix during SOS reaction when DNA replication is slowed down at the replication fork. DNA polymerase II, IV and V are translation polymerases.
• DNA Polymerase V is likewise engaged with translation synthesis during 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 synthesize primers. The smaller subunit has a primase activity. The biggest subunit has polymerization 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 DNA 

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.

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.

Primase

DNA primase is an enzyme whose continual activity is required at the DNA replication fork. They catalyze 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 synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long.

Endonucleases

These enzymes catalyze 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.

Single-Strand Binding Proteins

These proteins stabilize single-stranded DNA regions, preventing them from re-forming double-stranded structures.

DNA Replication Process in Prokaryotes

In prokaryotes, such as bacteria, DNA replication is a highly organised process that ensures the duplication of genetic material. The DNA replication in prokaryotes takes place in the following way:

• DNA strands unwind at the origin of replication and helicase enzyme facilitates the separation of DNA strands, leading to the formation of replication forks.
• Single-strand binding proteins coverthe DNA around the replication fork, preventing it from rewinding.
• Topoisomerase prevents excessive supercoiling of DNA during replication and primase synthesizes RNA primers that are complementary to the DNA strand.
• DNA polymerase III begins adding nucleotides to the end of the RNA primers.
• Leading and lagging strands undergo elongation.
• DNA Polymerase I removes primers and fills gaps, followed by sealing the gaps using ligase.

DNA Replication in Eukaryotes

The DNA replication in eukaryotes such as animals, plants, and fungi, is almost similar to the DNA replication in prokaryotes but is a more complex and regulated process that ensures the accurate duplication of genetic information. In eukaryotes, there are multiple origins of replication present and in eukaryotes, the polymerisation process is carried out by the enzyme Pol δ, whereas in prokaryotes it is carried out by DNA Pol III.

Importance of DNA Replication

The importance of DNA Replication is as follows:

• DNA replication helps in transfer of genetic information from one generation to the next during cell division, that enables inheritance of traits, characteristics, and maintains genetic diversity.
• DNA replication is important for repairing damaged DNA.
• DNA replication is essential for cell division, that 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.