Description

DNA replication is a fundamental process that occurs in all living organisms, ensuring that each new cell has an identical copy of the organism's genetic information. This complex, highly regulated process is essential for growth, development and tissue repair. DNA replication follows a semi-conservative model, where each of the two strands of the original DNA molecule serves as a template for the formation of a new complementary strand. The semi-conservative nature of replication was confirmed by the Meselson-Stahl experiment in 1958, which demonstrated that each new DNA molecule consists of one original strand and one newly synthesized strand. Replication begins at specific locations on the DNA molecule known as origins of replication. In bacteria, which have circular DNA, there is usually a single origin, while in eukaryotic cells, which have linear chromosomes, there are multiple origins of replication on each chromosome to expedite the replication process. These origins are recognized by certain proteins that initiate replication and start the unwinding of the DNA double helix. The unwinding of DNA at the origin is the first step in replication, facilitated by an enzyme called helicase. Helicase breaks the hydrogen bonds between the complementary bases, causing the two strands of the DNA helix to separate and create a Y-shaped structure known as the replication fork. This unwound section of DNA allows other enzymes involved in replication to access the template strands. To prevent the unwound DNA strands from reannealing or forming secondary structures, Single-Strand Binding Proteins (SSBs) bind to each strand, stabilizing them and maintaining the replication fork open for efficient replication. As helicase unwinds the DNA, it introduces positive supercoiling ahead of the replication fork. To alleviate this tension and prevent the DNA from becoming too tightly coiled, another enzyme called topoisomerase cuts the DNA, allows it to unwind and then reseals it, thus reducing the stress caused by supercoiling.

Exonuclease activity

Once the DNA strands are separated, DNA synthesis begins with the help of an enzyme called primase, which synthesizes a short RNA primer. This primer is necessary because DNA polymerases, the enzymes that add nucleotides to form the new DNA strand, cannot initiate synthesis on their own. They can only add nucleotides to an existing strand of nucleic acid, hence the need for the RNA primer. In the leading strand, which is oriented in the 3’ to 5’ direction relative to the direction of replication, DNA polymerase III (in prokaryotes) or DNA polymerase δ (in eukaryotes) can continuously add nucleotides to the 3’ end of the primer, synthesizing the new strand in a 5’ to 3’ direction without interruption. This continuous synthesis allows the leading strand to be replicated smoothly and efficiently as the replication fork progresses. However, DNA replication on the lagging strand is more complex due to its orientation in the 5’ to 3’ direction relative to the direction of replication, which is opposite to the direction of the replication fork. Since DNA polymerase can only synthesize DNA in the 5’ to 3’ direction, the lagging strand must be synthesized discontinuously in short segments called Okazaki fragments. Primase periodically lays down RNA primers along the lagging strand and DNA polymerase III (in prokaryotes) or DNA polymerase δ (in eukaryotes) extends each primer, creating these Okazaki fragments. However, there still remains a gap between the adjacent Okazaki fragments. This gap is sealed by the enzyme DNA ligase, which creates a phosphodiester bond between the 3’ hydroxyl end of one fragment and the 5’ phosphate end of the adjacent fragment, forming a continuous strand of DNA on the lagging strand. Through this coordinated series of events, both the leading and lagging strands are replicated, ensuring that each daughter molecule of DNA contains one old and one new strand, in accordance with the semi-conservative model of replication. DNA replication is an incredibly accurate process, but errors can still occur. DNA polymerases have a proofreading function, which helps maintain the high fidelity of replication. During synthesis, DNA polymerase checks each nucleotide it adds to ensure that it is complementary to the template strand. If an incorrect nucleotide is added, the polymerase can remove it through a 3’ to 5’ exonuclease activity and replace it with the correct nucleotide. This proofreading function significantly reduces the error rate of DNA replication. Additionally, after replication is complete, other cellular mechanisms, such as mismatch repair, can further correct any errors that might have escaped the proofreading process, preserving the integrity of the genetic information.

Disease prevention

In eukaryotic cells, DNA replication faces unique challenges due to the linear nature of chromosomes. At the ends of linear chromosomes, there is a problem known as the end-replication problem. DNA polymerase cannot fully replicate the very ends of the chromosomes, which could lead to a gradual shortening of chromosomes with each cell division. To counter this, eukaryotic chromosomes have repetitive sequences called telomeres at their ends, which protect essential genes from being eroded. An enzyme called telomerase extends these telomeres by adding repetitive nucleotide sequences, allowing cells to divide without losing important genetic information. Telomerase is highly active in germ cells, stem cells and some cancer cells, contributing to their ability to divide indefinitely, whereas in most somatic cells, telomerase activity is low or absent, leading to progressive telomere shortening and cellular aging. The regulation of DNA replication is tightly controlled to ensure that each segment of DNA is replicated only once per cell cycle. In eukaryotes, replication occurs during the S phase of the cell cycle and various checkpoints ensure that the DNA is fully replicated and undamaged before the cell proceeds to mitosis. The initiation of replication is regulated by a complex of proteins known as the Origin Recognition Complex (ORC), which binds to origins of replication and recruit’s additional factors to form a prereplication complex.